Transgenic plant with increased stress tolerance and yield

ABSTRACT

Polynucleotides are disclosed which are capable of enhancing growth, yield under water-limited conditions, and/or increased tolerance to an environmental stress of a plant transformed to contain such polynucleotides. Also provided are methods of using such polynucleotides and transgenic plants and agricultural products, including seeds, containing such polynucleotides as transgenes.

This application is a national stage application under 35 U.S.C. §371 ofPCT/EP2008/053382, filed Mar. 20, 2008, which claims benefit of U.S.provisional application Nos. 60/896,505, filed Mar. 23, 2007. The entirecontents of each of the above-identified applications are incorporatedherein by reference.

Please add the abstract as set forth in the attached supplemental sheetas a separate page.

FIELD OF THE INVENTION

This invention relates generally to transgenic plants which overexpressnucleic acid sequences encoding polypeptides capable of conferringincreased stress tolerance and consequently, increased plant growth andcrop yield, under normal or abiotic stress conditions. Additionally, theinvention relates to novel isolated nucleic acid sequences encodingpolypeptides that confer upon a plant increased tolerance under abioticstress conditions, and/or increased plant growth and/or increased yieldunder normal or abiotic stress conditions.

BACKGROUND OF THE INVENTION

Abiotic environmental stresses, such as drought, salinity, heat, andcold, are major limiting factors of plant growth and crop yield. Cropyield is defined herein as the number of bushels of relevantagricultural product (such as grain, forage, or seed) harvested peracre. Crop losses and crop yield losses of major crops such as soybean,rice, maize (corn), cotton, and wheat caused by these stresses representa significant economic and political factor and contribute to foodshortages in many underdeveloped countries.

Water availability is an important aspect of the abiotic stresses andtheir effects on plant growth. Continuous exposure to drought conditionscauses major alterations in the plant metabolism which ultimately leadto cell death and consequently to yield losses. Because high saltcontent in some soils results in less water being available for cellintake, high salt concentration has an effect on plants similar to theeffect of drought on plants. Additionally, under freezing temperatures,plant cells lose water as a result of ice formation within the plant.Accordingly, crop damage from drought, heat, salinity, and cold stress,is predominantly due to dehydration.

Because plants are typically exposed to conditions of reduced wateravailability during their life cycle, most plants have evolvedprotective mechanisms against dessication caused by abiotic stresses.However, if the severity and duration of dessication conditions are toogreat, the effects on development, growth, plant size, and yield of mostcrop plants are profound. Developing plants efficient in water use istherefore a strategy that has the potential to significantly improvehuman life on a worldwide scale.

Traditional plant breeding strategies are relatively slow and requireabiotic stress-tolerant founder lines for crossing with other germplasmto develop new abiotic stress-resistant lines. Limited germplasmresources for such founder lines and incompatibility in crosses betweendistantly related plant species represent significant problemsencountered in conventional breeding. Breeding for tolerance has beenlargely unsuccessful.

Many agricultural biotechnology companies have attempted to identifygenes that could confer tolerance to abiotic stress responses, in aneffort to develop transgenic abiotic stress-tolerant crop plants.Although some genes that are involved in stress responses or water useefficiency in plants have been characterized, the characterization andcloning of plant genes that confer stress tolerance and/or water useefficiency remains largely incomplete and fragmented. To date, successat developing transgenic abiotic stress-tolerant crop plants has beenlimited, and no such plants have been commercialized.

In order to develop transgenic abiotic stress-tolerant crop plants, itis necessary to assay a number of parameters in model plant systems,greenhouse studies of crop plants, and in field trials. For example,water use efficiency (WUE), is a parameter often correlated with droughttolerance. Studies of a plant's response to desiccation, osmotic shock,and temperature extremes are also employed to determine the plant'stolerance or resistance to abiotic stresses. When testing for the impactof the presence of a transgene on a plant's stress tolerance, theability to standardize soil properties, temperature, water and nutrientavailability and light intensity is an intrinsic advantage of greenhouseor plant growth chamber environments compared to the field.

WUE has been defined and measured in multiple ways. One approach is tocalculate the ratio of whole plant dry weight, to the weight of waterconsumed by the plant throughout its life. Another variation is to use ashorter time interval when biomass accumulation and water use aremeasured. Yet another approach is to use measurements from restrictedparts of the plant, for example, measuring only aerial growth and wateruse. WUE also has been defined as the ratio of CO₂ uptake to water vaporloss from a leaf or portion of a leaf, often measured over a very shorttime period (e.g. seconds/minutes). The ratio of ¹³C/¹²C fixed in planttissue, and measured with an isotope ratio mass-spectrometer, also hasbeen used to estimate WUE in plants using C₃ photosynthesis.

An increase in WUE is informative about the relatively improvedefficiency of growth and water consumption, but this information takenalone does not indicate whether one of these two processes has changedor both have changed. In selecting traits for improving crops, anincrease in WUE due to a decrease in water use, without a change ingrowth would have particular merit in an irrigated agricultural systemwhere the water input costs were high. An increase in WUE driven mainlyby an increase in growth without a corresponding jump in water use wouldhave applicability to all agricultural systems. In many agriculturalsystems where water supply is not limiting, an increase in growth, evenif it came at the expense of an increase in water use (i.e. no change inWUE), could also increase yield. Therefore, new methods to increase bothWUE and biomass accumulation are required to improve agriculturalproductivity.

Concomitant with measurements of parameters that correlate with abioticstress tolerance are measurements of parameters that indicate thepotential impact of a transgene on crop yield. For forage crops likealfalfa, silage corn, and hay, the plant biomass correlates with thetotal yield. For grain crops, however, other parameters have been usedto estimate yield, such as plant size, as measured by total plant dryweight, above-ground dry weight, above-ground fresh weight, leaf area,stem volume, plant height, rosette diameter, leaf length, root length,root mass, tiller number, and leaf number. Plant size at an earlydevelopmental stage will typically correlate with plant size later indevelopment. A larger plant with a greater leaf area can typicallyabsorb more light and carbon dioxide than a smaller plant and thereforewill likely gain a greater weight during the same period. This is inaddition to the potential continuation of the micro-environmental orgenetic advantage that the plant had to achieve the larger sizeinitially. There is a strong genetic component to plant size and growthrate, and so for a range of diverse genotypes plant size under oneenvironmental condition is likely to correlate with size under another.In this way a standard environment is used to approximate the diverseand dynamic environments encountered at different locations and times bycrops in the field.

Harvest index, the ratio of seed yield to above-ground dry weight, isrelatively stable under many environmental conditions and so a robustcorrelation between plant size and grain yield is possible. Plant sizeand grain yield are intrinsically linked, because the majority of grainbiomass is dependent on current or stored photosynthetic productivity bythe leaves and stem of the plant. Therefore, selecting for plant size,even at early stages of development, has been used as to screen forplants that may demonstrate increased yield when exposed to fieldtesting. As with abiotic stress tolerance, measurements of plant size inearly development, under standardized conditions in a growth chamber orgreenhouse, are standard practices to measure potential yield advantagesconferred by the presence of a transgene.

There is a need, therefore, to identify additional genes expressed instress tolerant plants and/or plants that are efficient in water usethat have the capacity to confer stress tolerance and/or increased wateruse efficiency to the host plant and to other plant species. Newlygenerated stress tolerant plants and/or plants with increased water useefficiency will have many advantages, such as an increased range inwhich the crop plants can be cultivated, by for example, decreasing thewater requirements of a plant species. Other desirable advantagesinclude increased resistance to lodging, the bending of shoots or stemsin response to wind, rain, pests, or disease.

SUMMARY OF THE INVENTION

The present inventors have discovered that transforming a plant withcertain polynucleotides results in enhancement of the plant's growthand/or response to environmental stress, and accordingly the yield ofthe agricultural products of the plant is increased, when thepolynucleotides are present in the plant as transgenes. Thepolynucleotides capable of mediating such enhancements have beenisolated from Physcomitrella patens, Brassica napus, Zea mays, Linumusitatissimum, Oryza satvia, Glycine max, or Triticum aestivum and arelisted in Table 1, and the sequences thereof are set forth in theSequence Listing as indicated in Table 1.

TABLE 1 Polynucleotide Amino acid Gene Name Gene ID Organism SEQ ID NOSEQ ID NO PpTPT-1 EST 214 P. patens 1 2 PpCDC2-1 EST 280 P. patens 3 4PpLRP-1 EST 298a P. patens 5 6 EST 298b P. patens 55 56 EST 298c P.patens 57 58 PpRBP-1 EST 300 P. patens 7 8 PpPD-1 EST 362 P. patens 9 10PpMSC-1 EST 378 P. patens 11 12 PpMBP-1 EST 398 P. patens 13 14 PpAK-1EST 407 P. patens 15 16 PpZF-6 EST 458 P. patens 17 18 PpCDK-1 EST 479P. patens 19 20 PpZF-7 EST 520 P. patens 21 22 PpMFP-1 EST 544 P. patens23 24 PpLRP-2 EST 545 P. patens 25 26 PpPPK-1 EST 549 P. patens 27 28PpSRP-1 EST 554 P. patens 29 30 PpCBL-1 EST 321 P. patens 31 32 PpCBL-2EST 416 P. patens 33 34 PpHD-1 EST 468 P. patens 35 36 BnHD-1 BN51361834B. napus 37 38 BnHD-2 BN50000854 B. napus 39 40 ZmHD-1 ZM59324542 Z.mays 41 42 LuHD-1 LU61552369 L. usitatissimum 43 44 OsHD-1 OS34631911 O.sativa 45 46 GmHD-1 GM59700314 G. max 47 48 GmHD-2 GM49753757 G. max 4950 GmHD-3 GM50270592 G. max 51 52 TaHD-1 TA60089198 T. aestivum 53 54

In one embodiment, the invention provides a transgenic plant transformedwith an expression cassette comprising an isolated polynucleotideencoding a tRNA 2′-phosphotransferase.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a cell division control protein kinase.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a leucine-rich repeat protein.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a Ran-binding protein.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a plastid division protein.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a mitochondrial substrate carrier protein.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a MADS-box protein.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolunucleotide encoding an adenosine kinase-1 protein.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a zinc finger-6 protein.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a cyclin-dependent kinase regulatory subunitprotein.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a zinc finger-7 protein.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a MAR-binding protein.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an polynucleotideencoding a leucine rich repeat receptor protein.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an polynucleotideencoding a phytochrome protein kinase protein.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an polynucleotideencoding a synaptobrevin protein.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an polynucleotideencoding a calcineurin B protein.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an polynucleotideencoding a caleosin protein.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an polynucleotideencoding a histone deacetylase protein.

In a further embodiment, the invention concerns a seed produced by thetransgenic plant of the invention, wherein the seed is true breeding fora transgene comprising the polynucleotide described above. Plantsderived from the seed of the invention demonstrate increased toleranceto an environmental stress, and/or increased plant growth, and/orincreased yield, under normal or stress conditions as compared to a wildtype variety of the plant.

In a still another aspect, the invention concerns products produced byor from the transgenic plants of the invention, their plant parts, ortheir seeds, such as a foodstuff, feedstuff, food supplement, feedsupplement, cosmetic or pharmaceutical.

The invention further provides the isolated polynucleotides identifiedin Table 1 or in Table 2 below, and polypeptides identified in Table 1.The invention is also embodied in recombinant vector comprising anisolated polynucleotide of the invention.

In yet another embodiment, the invention concerns a method of producingthe aforesaid transgenic plant, wherein the method comprisestransforming a plant cell with an expression vector comprising anisolated polynucleotide of the invention, and generating from the plantcell a transgenic plant that expresses the polypeptide encoded bythepolynucleotide. Expression of the polypeptide in the plant results inincreased tolerance to an environmental stress, and/or growth, and/oryield under normal and/or stress conditions as compared to a wild typevariety of the plant.

In still another embodiment, the invention provides a method ofincreasing a plant's tolerance to an environmental stress, and/orgrowth, and/or yield. The method comprises the steps of transforming aplant cell with an expression cassette comprising an isolatedpolynucleotide of the invention, and generating a transgenic plant fromthe plant cell, wherein the transgenic plant comprises thepolynucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating the phylogenetic relationship among thedisclosed PpHD-1 (SEQ ID NO:36), BnHD-1 (SEQ ID NO:38), BnHD-2 (SEQ IDNO:40), ZmHD-1 (SEQ ID NO:42), LuHD-1 (SEQ ID NO:44), OsHD-1 (SEQ IDNO:46), GmHD-1 (SEQ ID NO:48), GmHD-2 (SEQ ID NO:50), GmHD-3 (SEQ IDNO:52), and TaHD-1 (SEQ ID NO:54) amino acid sequences. The diagram wasgenerated using Align X of Vector NTI.

FIG. 2 shows an alignment of the disclosed amino acids sequences: PpHD-1(SEQ ID NO:36), BnHD-1 (SEQ ID NO:38), BnHD-2 (SEQ ID NO:40), ZmHD-1(SEQ ID NO:42), LuHD-1 (SEQ ID NO:44), OsHD-1 (SEQ ID NO:46), GmHD-1(SEQ ID NO:48), GmHD-2 (SEQ ID NO:50), GmHD-3 (SEQ ID NO:52), and TaHD-1(SEQ ID NO:54). The alignment was generated using Align X of Vector NTI.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Throughout this application, various publications are referenced. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains. The terminology usedherein is for the purpose of describing specific embodiments only and isnot intended to be limiting. As used herein, “a” or “an” can mean one ormore, depending upon the context in which it is used. Thus, for example,reference to “a cell” can mean that at least one cell can be used.

In one embodiment, the invention provides a transgenic plant thatoverexpresses an isolated polynucleotide identified in Table 1, or ahomolog thereof. The transgenic plant of the invention demonstrates anincreased tolerance to an environmental stress as compared to a wildtype variety of the plant. The overexpression of such isolated nucleicacids in the plant may optionally result in an increase in plant growthor in yield of associated agricultural products, under normal or stressconditions, as compared to a wild type variety of the plant. Withoutwishing to be bound by any theory, the increased tolerance to anenvironmental stress, increased growth, and/or increased yield of atransgenic plant of the invention is believed to result from an increasein water use efficiency of the plant.

As defined herein, a “transgenic plant” is a plant that has been alteredusing recombinant DNA technology to contain an isolated nucleic acidwhich would otherwise not be present in the plant. As used herein, theterm “plant” includes a whole plant, plant cells, and plant parts. Plantparts include, but are not limited to, stems, roots, ovules, stamens,leaves, embryos, meristematic regions, callus tissue, gametophytes,sporophytes, pollen, microspores, and the like. The transgenic plant ofthe invention may be male sterile or male fertile, and may furtherinclude transgenes other than those that comprise the isolatedpolynucleotides described herein.

As used herein, the term “variety” refers to a group of plants within aspecies that share constant characteristics that separate them from thetypical form and from other possible varieties within that species.While possessing at least one distinctive trait, a variety is alsocharacterized by some variation between individuals within the variety,based primarily on the Mendelian segregation of traits among the progenyof succeeding generations. A variety is considered “true breeding” for aparticular trait if it is genetically homozygous for that trait to theextent that, when the true-breeding variety is self-pollinated, asignificant amount of independent segregation of the trait among theprogeny is not observed. In the present invention, the trait arises fromthe transgenic expression of one or more isolated polynucleotidesintroduced into a plant variety. As also used herein, the term “wildtype variety” refers to a group of plants that are analyzed forcomparative purposes as a control plant, wherein the wild type varietyplant is identical to the transgenic plant (plant transformed with anisolated polynucleotide in accordance with the invention) with theexception that the wild type variety plant has not been transformed tocontain an isolated polynucleotide of the invention.

As defined herein, the term “nucleic acid” and “polynucleotide” areinterchangeable and refer to RNA or DNA that is linear or branched,single or double stranded, or a hybrid thereof. The term alsoencompasses RNA/DNA hybrids. An “isolated” nucleic acid molecule is onethat is substantially separated from other nucleic acid molecules whichare present in the natural source of the nucleic acid (i.e., sequencesencoding other polypeptides). For example, a cloned nucleic acid isconsidered isolated. A nucleic acid is also considered isolated if ithas been altered by human intervention, or placed in a locus or locationthat is not its natural site, or if it is introduced into a cell bytransformation. Moreover, an isolated nucleic acid molecule, such as acDNA molecule, can be free from some of the other cellular material withwhich it is naturally associated, or culture medium when produced byrecombinant techniques, or chemical precursors or other chemicals whenchemically synthesized. While it may optionally encompass untranslatedsequence located at both the 3′ and 5′ ends of the coding region of agene, it may be preferable to remove the sequences which naturally flankthe coding region in its naturally occurring replicon.

As used herein, the term “environmental stress” refers to a sub-optimalcondition associated with salinity, drought, nitrogen, temperature,metal, chemical, pathogenic, or oxidative stresses, or any combinationthereof. The terms “water use efficiency” and “WUE” refer to the amountof organic matter produced by a plant divided by the amount of waterused by the plant in producing it, i.e., the dry weight of a plant inrelation to the plant's water use. As used herein, the term “dry weight”refers to everything in the plant other than water, and includes, forexample, carbohydrates, proteins, oils, and mineral nutrients.

Any plant species may be transformed to create a transgenic plant inaccordance with the invention. The transgenic plant of the invention maybe a dicotyledonous plant or a monocotyledonous plant. For example andwithout limitation, transgenic plants of the invention may be derivedfrom any of the following diclotyledonous plant families: Leguminosae,including plants such as pea, alfalfa and soybean; Umbelliferae,including plants such as carrot and celery; Solanaceae, including theplants such as tomato, potato, aubergine, tobacco, and pepper;Cruciferae, particularly the genus Brassica, which includes plant suchas oilseed rape, beet, cabbage, cauliflower and broccoli); andArabidopsis thaliana; Compositae, which includes plants such as lettuce;Malvaceae, which includes cotton; Fabaceae, which includes plants suchas peanut, and the like. Transgenic plants of the invention may bederived from monocotyledonous plants, such as, for example, wheat,barley, sorghum, millet, rye, triticale, maize, rice, oats, switchgrass,miscanthus, and sugarcane. Transgenic plants of the invention are alsoembodied as trees such as apple, pear, quince, plum, cherry, peach,nectarine, apricot, papaya, mango, and other woody species includingconiferous and deciduous trees such as poplar, pine, sequoia, cedar,oak, willow, and the like. Especially preferred are Arabidopsisthaliana, Nicotiana tabacum, oilseed rape, soybean, corn (maize), wheat,linseed, potato and tagetes.

As shown in Table 1, one embodiment of the invention is a transgenicplant transformed with an expression cassette comprising an isolatedpolynucleotide encoding a tRNA 2′-phosphotransferase polypeptide. Inyeast, the RNA 2′-phosphotransferase Tpt1 protein is an essentialprotein that catalyzes the final step of tRNA splicing. Although thisfamily of proteins is conserved in eukaryotes, bacteria, and archaea,its function has only been well characterized in yeast. tRNA splicing isconserved in all three major kingdoms, but the mechanisms and enzymesinvolved differ. These differences leave the exact function of RNA2′-phoshotransferase proteins in plants unclear, although the enzymaticactivity has been demonstrated in tobacco nuclear extracts. All of theRNA 2′phosphotransferase family members contain a conserved core domain,exemplified by amino acids 98 to 287 of SEQ ID NO:2, and members fromEscherichia coli, Arabidopsis thaliana, Schizosaccharomyces pombe, andHomo sapiens are capable of complementing the Saccharomyces cerevisiaetpt1 mutant, indicating similarity of function.

The transgenic plant of this embodiment may comprise any polynucleotideencoding a tRNA 2′-phosphotransferase. Preferably, the transgenic plantof this embodiment comprises a polynucleotide encoding a tRNA2′-phosphotransferase having a sequence comprising amino acids 98 to 287of SEQ ID NO:2. More preferably, the transgenic plant of this embodimentcomprises a polynucleotide encoding a tRNA 2′-phosphotransferase havinga sequence comprising amino acids 1 to 323 of SEQ ID NO:2.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a cell division control 2 (CDC2) protein kinase.The CDC2 proteins belong to a specific family of cyclin-dependentkinases (CDKs) in plants commonly referred to as the CDKA family. All ofthe CDKA proteins contain a highly conserved core kinase domain with aPSTAIRE motif that is the principle site for cyclin interaction to formactive CDK-cyclin complexes. An exemplary PSTAIRE motif is representedas amino acids 4 to 287 of SEQ ID NO:4. The CDKA proteins are alsosubject to posttranslational modification. Phosphorylation of theconserved threonine 14 and tyrosine 15 positions inactivates the CDKA,and phosphorylation of the conserved threonine 161 position activatesthe CDKA. In yeast these CDKs are involved specifically in G1/S and G2/Mcontrols. In plants, CDKA's are proposed to function in both S and Mphase progression and to be involved in cell proliferation andmaintenance of cell division competence in differentiating tissues. InArabidopsis thaliana for example, a mutation of the CDKA1 gene leads tomale gametophytic lethality and impairs seed development by reducingseed size.

The transgenic plant of this embodiment may comprise any polynucleotideencoding a CDC2 protein kinase. Preferably, the transgenic plant of thisembodiment comprises a polynucleotide encoding a CDKA protein having asequence comprising amino acids 4 to 287 of SEQ ID NO:4. Morepreferably, the transgenic plant of this embodiment comprises apolynucleotide encoding a CDKA protein having a sequence comprisingamino acids 1 to 294 of SEQ ID NO:4.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a leucine-rich repeat (LRR) protein. LRRs aretypically found in proteins as 20 to 29 amino acids repeats, eachcontaining an 11 amino acid conserved region with the consensus sequenceof LXXLXLXXN/CXL with X as any amino acid and L as valine, leucine, orphenylalanine. The LRR protein of the present invention contains an LRRrepresented by amino acids 422 to 441 of SEQ ID NO:6. The generallyaccepted major function of LLRs is to provide a structural scaffold forthe formation of protein-protein interactions. LLR-containing proteinsare known to be involved in hormone-receptor interactions, enzymeinhibition, cell adhesion, celluar trafficking, plant diseaseresistance, and bacterial virulence.

The transgenic plant of this embodiment may comprise any polynucleotideencoding an LRR protein having a sequence comprising amino acids 422 to441 of SEQ ID NO:6. More preferably, the transgenic plant of thisembodiment comprises a polynucleotide encoding a LRR protein having asequence comprising amino acids 1 to 646 of SEQ ID NO:6.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a Ran-binding protein. Ran GTPase (RanGTP)proteins belong to a subfamily of small GTP-binding proteins that areinvolved in nucleocytoplasmic transport, and are involved in controllingnuclear functions throughout the cell cycle. The Ran binding proteins 1(RanBP1s) are cytoplasmic proteins that form a complex with the GTP formof RanGTP. The binding domain of RanBP1 s that interacts with RanGTP hasbeen identified and is represented by amino acids 51 to 172 of SEQ IDNO:8. The formation of this RanGTP-RanBP1 complex is key to promotingthe initial dissociation of RanGTP from transport factors that areexported from the nucleus to the cytoplasm.

The transgenic plant of this embodiment may comprise any polynucleotideencoding RanBP1 protein having a sequence comprising amino acids 51 to172 of SEQ ID NO:8. More preferably, the transgenic plant of thisembodiment comprises a polynucleotide encoding a RanBP1 protein having asequence comprising amino acids 1 to 213 of SEQ ID NO:8.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a plastid division protein. The FtsZ plastiddivision proteins are characterized by domains represented by aminoacids 139 to 332 of SEQ ID NO:10. The transgenic plant of thisembodiment may comprise any polynucleotide encoding a plastid divisionprotein having a sequence comprising amino acids 139 to 332 of SEQ IDNO:10. More preferably, the transgenic plant of this embodimentcomprises a polynucleotide encoding a plastid division protein having asequence comprising amino acids 1 to 490 of SEQ ID NO:10.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a mitochondrial substrate carrier protein. Themitochondrial substrate carrier proteins are characterized by domainsrepresented by amino acids 1 to 98 of SEQ ID NO:12. The transgenic plantof this embodiment may comprise any polynucleotide encoding amitochondrial substrate carrier protein having a sequence comprisingamino acids 1 to 98 of SEQ ID NO:12. More preferably, the transgenicplant of this embodiment comprises a polynucleotide encoding amitochondrial substrate carrier protein having a sequence comprisingamino acids 1 to 297 of SEQ ID NO:12.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a MADS-box protein. The DNA binding anddimerization domains of SRF-type transcription factors comprise MADS-boxdomains represented by amino acids 9 to 59 of SEQ ID NO:14. Thetransgenic plant of this embodiment may comprise any polynucleotideencoding an SRF-type transcription factor protein comprising a MADS-boxdomain having a sequence comprising amino acids 9 to 59 of SEQ ID NO:14.More preferably, the transgenic plant of this embodiment comprises apolynucleotide encoding a MADS-box protein having a sequence comprisingamino acids 1 to 187 of SEQ ID NO:14.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding an adenosine kinase-1 (ADK-1) protein. The pfkBfamily of carbohydrate kinases designated ADK-1 comprise domainsrepresented by amino acids 23 to 339 of SEQ ID NO:16. The transgenicplant of this embodiment may comprise any polynucleotide encoding anADK-1 protein comprising a domain having a sequence comprising aminoacids 23 to 339 of SEQ ID NO:16. More preferably, the transgenic plantof this embodiment comprises a polynucleotide encoding an ADK-1 proteinhaving a sequence comprising amino acids 1 to 343 of SEQ ID NO:16.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a zinc finger-6 (ZF-6) protein. These proteinscomprise an IBR domain represented by amino acids 210 to 272 of SEQ IDNO:18. The transgenic plant of this embodiment may comprise anypolynucleotide encoding a ZF-6 protein comprising a domain having asequence comprising amino acids 210 to 272 of SEQ ID NO:18. Morepreferably, the transgenic plant of this embodiment comprises apolynucleotide encoding a ZF-6 protein having a sequence comprisingamino acids 1 to 594 of SEQ ID NO:18.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a cyclin-dependent kinase regulatory subunit(CDK) protein. These proteins comprise a domain represented by aminoacids 1 to 72 of SEQ ID NO:20. The transgenic plant of this embodimentmay comprise any polynucleotide encoding a CDK protein comprising adomain having a sequence comprising amino acids 1 to 72 of SEQ ID NO:20.More preferably, the transgenic plant of this embodiment comprises apolynucleotide encoding a CDK protein having a sequence comprising aminoacids 1 to 91 of SEQ ID NO:20.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a zinc finger-7 (ZF-7) protein. These proteinscomprise a C₃HC4-type domain represented by amino acids 20 to 60 of SEQID NO:22. The transgenic plant of this embodiment may comprise anypolynucleotide encoding a ZF-7 protein comprising a domain having asequence comprising amino acids 20 to 60 of SEQ ID NO:22. Morepreferably, the transgenic plant of this embodiment comprises apolynucleotide encoding a ZF-7 protein having a sequence comprisingamino acids 1 to 347 of SEQ ID NO:22.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a MAR-binding protein. The transgenic plant ofthis embodiment comprises a polynucleotide encoding a MAR-bindingprotein having a sequence comprising amino acids 1 to 814 of SEQ IDNO:24.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an polynucleotideencoding a leucine rich repeat receptor protein kinase. The LRP-2protein of the present invention contains several LRRs, represented byamino acids 111 to 133 of SEQ ID NO:26, amino acids 135 to 158 of SEQ IDNO:26, amino acids 160 to 182 of SEQ ID NO:26, and amino acids 184 to207 of SEQ ID NO:26. The transgenic plant of this embodiment maycomprise any polynucleotide encoding an LRP-2 protein having a sequencecomprising amino acids 111 to 133 of SEQ ID NO:26, amino acids 135 to158 of SEQ ID NO:26, amino acids 160 to 182 of SEQ ID NO:26, and aminoacids 184 to 207 of SEQ ID NO:26. More preferably, the transgenic plantof this embodiment comprises a polynucleotide encoding a LRR proteinhaving a sequence comprising amino acids 1 to 251 of SEQ ID NO:26.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an polynucleotideencoding a phytochrome protein kinase protein. The transgenic plant ofthis embodiment comprises a polynucleotide encoding a phytochromeprotein kinase protein having a sequence comprising amino acids 1 to 689of SEQ ID NO:28.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an polynucleotideencoding a synaptobrevin-related protein.

These proteins comprise a synaptobrevin domain represented by aminoacids 127 to 215 of SEQ ID NO:30. The transgenic plant of thisembodiment may comprise any polynucleotide encoding asynaptobrevin-related protein comprising a domain having a sequencecomprising amino acids 127 to 215 of SEQ ID NO:30. More preferably, thetransgenic plant of this embodiment comprises a polynucleotide encodinga synaptobrevin-related protein having a sequence comprising amino acids1 to 222 of SEQ ID NO:30.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an polynucleotideencoding a calcineurin B protein. In plants, a family of proteins hasbeen found that are calcium sensor proteins with similarity to both theregulatory B subunit of calcineurin and neuronal calcium sensors ofanimals. These proteins have been termed calcineurin B-like proteins(CBL). These CBL proteins contain EF hand motifs that are structurallyimportant for calcium binding and interact specifically with a group ofSer/Thr protein kinases, designated as CBL-interacting protein kinases(CIPK). CIPKs most likely represent targets of calcium sensed andtransduced by CBL proteins.

Each EF hand consists of a loop of 12 amino acids flanked by two alphahelices, which binds a single calcium ion via the loop domain. Theseproteins have also been found to bind magnesium ions. Proteins with fourEF hand motifs usually have two structural domains, each formed by apair of EF hand motifs separated by a flexible linker. Binding of themetal ion to the EF hand protein leads to a conformational change thatexposes a hydrophobic surface, which binds to a target sequence. Many EFhand containing proteins also contain a myristoylation site at theN-terminus, with consensus sequence of MGXXXS/T, with X representing anyamino acid. Myristoylation at this site promotes protein-protein orprotein membrane interaction. This myristoylation site is not present inthe EST321 (SEQ ID NO:32) sequence, potentially indicating that theEST321 protein could belong to a different class of EF hand domaincontaining proteins.

The calcineurin B subunit protein of the present invention containsseveral EF hand motifs, represented by amino acids 37 to 65 of SEQ IDNO:32, amino acids 106 to 134 of SEQ ID NO:32, and amino acids 142 to170 of SEQ ID NO:32. The transgenic plant of this embodiment maycomprise any polynucleotide encoding a calcineurin B subunit proteinhaving a sequence comprising amino acids 37 to 65 of SEQ ID NO:32, aminoacids 106 to 134 of SEQ ID NO:32, and amino acids 142 to 170 of SEQ IDNO:32. More preferably, the transgenic plant of this embodimentcomprises a polynucleotide encoding a calcineurin B subunit proteinhaving a sequence comprising amino acids 1 to 182 of SEQ ID NO:32.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an polynucleotideencoding a caleosin-related protein. Caleosins are a family of proteinsare presumably modulated by calcium-binding and phosphorylation stateand are thought to be involved in fusion of membranes and oil bodies.These proteins contain several domains, an N-terminal region with asingle calcium ion binding EF-hand motif, a central hydrophobic regionwith a potential membrane anchor, and a C-terminal region with conservedprotein kinase phosphorylation sites. The presence of only a single EFhand motif is unusual for most EF hand containing proteins. It has beenpostulated that this single EF hand domain may interact with themembrane surface or another protein in order to form the coordinateddouble EF hand domain interaction found in most other EF hand proteins.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an polynucleotideencoding a caleosin-related protein. These proteins comprise a caleosindomain represented by amino acids 26 to 229 of SEQ ID NO:34. Thetransgenic plant of this embodiment may comprise any polynucleotideencoding a caleosin-related protein comprising a domain having asequence comprising amino acids 26 to 229 of SEQ ID NO:34. Morepreferably, the transgenic plant of this embodiment comprises apolynu-cleotide encoding a caleosin-related protein having a sequencecomprising amino acids 1 to 239 of SEQ ID NO:34.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an polynucleotideencoding a histone deacetylase protein. Nucleosomes consist of histonesand DNA, which are essential for packaging DNA into chromosomes. Lysineat the N-terminal ends of core histones are the predominant sites foracetylation and methylation, and histone deacetylases catalyze theremoval of the acetyl group from these lysine side chains. Active genesare preferentially associated with highly acetylated histones andinactive genes are associated with hypoacetylated histones. Acetylationresults in charge neutralization of histones and weakens histone/DNAcontacts. In plants, histone hyperacetylation is correlated with geneactivity.

Histones are found to be associated with large multisubunit complexes.Three distinct families of histone deacytelases are found in plants, theRPD3/HDA family, SIR2 family, and the plant specific HD2 family. TheRPD3/HDA1 family is found in all eukaryotic organisms, and memberspossess a complete histone deacetylase domain. Some histone deacetylaseproteins possess unique regions outside the histone deacetylase domainthat may be important for function and/or specificity of these proteins.

The histone deacetylases of the present invention are characterized bythe following domains: from amino acids 6 to 318 of SEQ ID NO:36; fromamino acids 6 to 318 of SEQ ID NO:38; from amino acids 20 to 332 of SEQID NO:40; from amino acids 8 to 322 of SEQ ID NO:42; from amino acids 6to 318 of SEQ ID NO:44; from amino acids 23 to 333 of SEQ ID NO:46; fromamino acids 8 to 321 of SEQ ID NO:48; from amino acids 6 to 318 of SEQID NO:50; from amino acids 56 to 382 of SEQ ID NO:52; and from aminoacids 23 to 333 of SEQ ID NO:54.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an polynucleotideencoding a histone deacetylase protein. The transgenic plant of thisembodiment may comprise any polynucleotide encoding a histonedeacetylase protein comprising a domain having a sequence selected fromthe group consisting of amino acids 6 to 318 of SEQ ID NO:36; aminoacids 6 to 318 of SEQ ID NO:38; amino acids 20 to 332 of SEQ ID NO:40;amino acids 8 to 322 of SEQ ID NO:42; amino acids 6 to 318 of SEQ IDNO:44; amino acids 23 to 333 of SEQ ID NO:46; amino acids 8 to 321 ofSEQ ID NO:48; amino acids 6 to 318 of SEQ ID NO:50; amino acids 56 to382 of SEQ ID NO:52; and amino acids 23 to 333 of SEQ ID NO:54. Morepreferably, the transgenic plant of this embodiment comprises apolynucleotide encoding a histone deacteylase protein selected from thegroup consisting of a protein having a sequence comprising amino acids 1to 431 of SEQ ID NO:36; a protein having a sequence comprising aminoacids 1 to 426 of SEQ ID NO:38; a protein having a sequence comprisingamino acids 1 to 470 of SEQ ID NO:40; a protein having a sequencecomprising amino acids 1 to 363 of SEQ ID NO:42; a protein having asequence comprising amino acids 1 to 429 of SEQ ID NO:44; a proteinhaving a sequence comprising amino acids 1 to 518 of SEQ ID NO:46; aprotein having a sequence comprising amino acids 1 to 334 of SEQ IDNO:48; a protein having a sequence comprising amino acids 1 to 429 ofSEQ ID NO:50; a protein having a sequence comprising amino acids 1 to417 of SEQ ID NO:52; and a protein having a sequence comprising aminoacids 1 to 519 of SEQ ID NO:54.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a leucine-rich repeat (LRR) protein. LRRs aretypically found in proteins as 20 to 29 amino acids repeats, eachcontaining an 11 amino acid conserved region with the consensus sequenceof LXXLXLXXN/CXL with X as any amino acid and L as valine, leucine, orphenylalanine. The LRR protein of the present invention contains an LRRrepresented by amino acids 422 to 441 of SEQ ID NO:56. The generallyaccepted major function of LLRs is to provide a structural scaffold forthe formation of protein-protein interactions. LLR-containing proteinsare known to be involved in hormone-receptor interactions, enzymeinhibition, cell adhesion, celluar trafficking, plant diseaseresistance, and bacterial virulence.

The transgenic plant of this embodiment may comprise any polynucleotideencoding an LRR protein having a sequence comprising amino acids 422 to441 of SEQ ID NO:56. More preferably, the transgenic plant of thisembodiment comprises a polynucleotide encoding a LRR protein having asequence comprising amino acids 1 to 698 of SEQ ID NO:56.

In another embodiment, the invention provides a transgenic planttransformed with an expression cassette comprising an isolatedpolynucleotide encoding a leucine-rich repeat (LRR) protein. LRRs aretypically found in proteins as 20 to 29 amino acids repeats, eachcontaining an 11 amino acid conserved region with the consensus sequenceof LXXLXLXXN/CXL with X as any amino acid and L as valine, leucine, orphenylalanine. The LRR protein of the present invention contains an LRRrepresented by amino acids 422 to 441 of SEQ ID NO:58. The generallyaccepted major function of LLRs is to provide a structural scaffold forthe formation of protein-protein interactions. LLR-containing proteinsare known to be involved in hormone-receptor interactions, enzymeinhibition, cell adhesion, celluar trafficking, plant diseaseresistance, and bacterial virulence.

The transgenic plant of this embodiment may comprise any polynucleotideencoding an LRR protein having a sequence comprising amino acids 422 to441 of SEQ ID NO:58. More preferably, the transgenic plant of thisembodiment comprises a polynucleotide encoding a LRR protein having asequence comprising amino acids 1 to 665 of SEQ ID NO:58.

The invention further provides a seed produced by a transgenic plantexpressing polynucleotide listed in Table 1, wherein the seed containsthe polynucleotide, and wherein the plant is true breeding for increasedgrowth and/or yield under normal and/or stress conditions and/orincreased tolerance to an environmental stress as compared to a wildtype variety of the plant. The invention also provides a productproduced by or from the transgenic plants expressing the polynucleotide,their plant parts, or their seeds. The product can be obtained usingvarious methods well known in the art. As used herein, the word“product” includes, but not limited to, a foodstuff, feedstuff, a foodsupplement, feed supplement, cosmetic or pharmaceutical. Foodstuffs areregarded as compositions used for nutrition or for supplementingnutrition. Animal feedstuffs and animal feed supplements, in particular,are regarded as foodstuffs. The invention further provides anagricultural product produced by any of the transgenic plants, plantparts, and plant seeds. Agricultural products include, but are notlimited to, plant extracts, proteins, amino acids, carbohydrates, fats,oils, polymers, vitamins, and the like.

In a preferred embodiment, an isolated polynucleotide of the inventioncomprises a polynucleotide having a sequence selected from the groupconsisting of the polynucleotide sequences listed in Table 1. Thesepolynucleotides may comprise sequences of the coding region, as well as5′ untranslated sequences and 3′ untranslated sequences. Table 2describes potential start and end positions of the coding regions of theP. patens polynucleotides of the invention, and alternative open readingframes that may be present in the sense or antisense strands of thesepolynucleotides. Alternatively, the polynucleotides of the invention cancomprise only the coding region of the nucleotide sequences listed inTable 1, as indicated in Table 2, or the polynucleotides can containwhole genomic fragments isolated from genomic DNA. Thus the invention isalso embodied as an isolated polynucleotide having a sequence selectedfrom the group consisting of the sequences listed in Table 1 or Table 2.

TABLE 2 Gene Name GENE ID SEQ ID NO ORFs Orf Number Strand StartPosition End Position PpTPT-1 EST 214 1 1 1 sense 113 1104 PpCDC2-1 EST280 3 2 1 sense 37 921 PpCDC2-1 EST 280 3 2 2 antisense 380 42 PpLRP-1EST 298a 5 1 1 sense 144 2084 EST 298b 55 1 1 sense 143 2236 EST 298c 571 1 sense 1 1998 PpRBP-1 EST 300 7 1 1 sense 55 696 PpPD-1 EST 362 9 2 1sense 47 1519 PpPD-1 EST 362 9 2 2 antisense 1197 604 PpMSC-1 EST 378 112 1 sense 453 1346 PpMSC-1 EST 378 11 2 2 antisense 1314 1027 PpMBP-1EST 398 13 1 1 sense 33 878 PpAK-1 EST 407 15 3 1 sense 25 1056 PpAK-1EST 407 15 3 2 sense 381 632 PpAK-1 EST 407 15 3 3 antisense 506 270PpZF-6 EST 458 17 1 1 sense 126 1910 PpCDK-1 EST 479 19 2 1 sense 248523 PpCDK-1 EST 479 19 2 2 antisense 304 104 PpZF-7 EST 520 21 2 1 sense276 1319 PpZF-7 EST 520 21 2 2 sense 583 813 PpMFP-1 EST 544 23 1 1sense 127 2571 PpLRP-2 EST 545 25 3 1 sense 225 980 PpLRP-2 EST 545 25 32 sense 416 694 PpLRP-2 EST 545 25 3 3 antisense 469 167 PpPPK-1 EST 54927 1 1 sense 145 2214 PpSRP-1 EST 554 29 1 1 sense 20 688 PpCBL-1 EST321 31 2 1 sense 43 591 PpCBL-1 EST 321 31 2 2 sense 803 1171 PpCBL-2EST 416 33 1 1 sense 16 735 PpHD-1 EST 468 35 2 1 sense 166 1461 PpHD-1EST 468 35 2 2 antisense 420 175

A polynucleotide of the invention can be isolated using standardmolecular biology techniques and the sequence information providedherein. For example, P. patens cDNAs of the invention were isolated froma P. patens library using a portion of the sequence disclosed herein.Synthetic oligonucleotide primers for polymerase chain reactionamplification can be designed based upon the nucleotide sequence shownin Table 1. A nucleic acid molecule of the invention can be amplifiedusing cDNA or, alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid molecule so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to the nucleotide sequenceslisted in Table 1 can be prepared by standard synthetic techniques,e.g., using an automated DNA synthesizer.

“Homologs” are defined herein as two nucleic acids or polypeptides thathave similar, or substantially identical, nucleotide or amino acidsequences, respectively. Homologs include allelic variants, analogs, andorthologs, as defined below. As used herein, the term “analogs” refersto two nucleic acids that have the same or similar function, but thathave evolved separately in unrelated organisms. As used herein, the term“orthologs” refers to two nucleic acids from different species, but thathave evolved from a common ancestral gene by speciation. The termhomolog further encompasses nucleic acid molecules that differ from oneof the nucleotide sequences shown in Table 1 due to degeneracy of thegenetic code and thus encode the same polypeptide. As used herein, a“naturally occurring” nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural polypeptide).

To determine the percent sequence identity of two amino acid sequences(e.g., one of the polypeptide sequences of Table 1 and a homologthereof), the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of one polypeptide foroptimal alignment with the other polypeptide or nucleic acid). The aminoacid residues at corresponding amino acid positions are then compared.When a position in one sequence is occupied by the same amino acidresidue as the corresponding position in the other sequence then themolecules are identical at that position. The same type of comparisoncan be made between two nucleic acid sequences.

Preferably, the isolated amino acid homologs, analogs, and orthologs ofthe polypeptides of the present invention are at least about 50-60%,preferably at least about 60-70%, and more preferably at least about70-75%, 75-80%, 80-85%, 85-90%, or 90-95%, and most preferably at leastabout 96%, 97%, 98%, 99%, or more identical to an entire amino acidsequence identified in Table 1. In another preferred embodiment, anisolated nucleic acid homolog of the invention comprises a nucleotidesequence which is at least about 40-60%, preferably at least about60-70%, more preferably at least about 70-75%, 75-80%, 80-85%, 85-90%,or 90-95%, and even more preferably at least about 95%, 96%, 97%, 98%,99%, or more identical to a nucleotide sequence shown in Table 1 orTable 2.

For the purposes of the invention, the percent sequence identity betweentwo nucleic acid or polypeptide sequences is determined using the VectorNTI 9.0 (PC) software package (Invitrogen, 1600 Faraday Ave., Carlsbad,Calif. 92008). A gap opening penalty of 15 and a gap extension penaltyof 6.66 are used for determining the percent identity of two nucleicacids. A gap opening penalty of 10 and a gap extension penalty of 0.1are used for determining the percent identity of two polypeptides. Allother parameters are set at the default settings. For purposes of amultiple alignment (Clustal W algorithm), the gap opening penalty is 10,and the gap extension penalty is 0.05 with blosum62 matrix. It is to beunderstood that for the purposes of determining sequence identity whencomparing a DNA sequence to an RNA sequence, a thymidine nucleotide isequivalent to a uracil nucleotide.

Nucleic acid molecules corresponding to homologs, analogs, and orthologsof the polypeptides listed in Table 1 can be isolated based on theiridentity to said polypeptides, using the polynucleotides encoding therespective polypeptides or primers based thereon, as hybridizationprobes according to standard hybridization techniques under stringenthybridization conditions. As used herein with regard to hybridizationfor DNA to a DNA blot, the term “stringent conditions” refers tohybridization overnight at 60° C. in 10× Denhart's solution, 6×SSC, 0.5%SDS, and 100 μg/ml denatured salmon sperm DNA. Blots are washedsequentially at 62° C. for 30 minutes each time in 3×SSC/0.1% SDS,followed by 1×SSC/0.1% SDS, and finally 0.1×SSC/0.1% SDS. As also usedherein, in a preferred embodiment, the phrase “stringent conditions”refers to hybridization in a 6×SSC solution at 65° C. In anotherembodiment, “highly stringent conditions” refers to hybridizationovernight at 65° C. in 10× Denhart's solution, 6×SSC, 0.5% SDS and 100μg/ml denatured salmon sperm DNA. Blots are washed sequentially at 65°C. for 30 minutes each time in 3×SSC/0.1% SDS, followed by 1×SSC/0.1%SDS, and finally 0.1×SSC/0.1% SDS. Methods for nucleic acidhybridizations are described in Meinkoth and Wahl, 1984, Anal. Biochem.138:267-284; well known in the art (see, for example, Current Protocolsin Molecular Biology, Chapter 2, Ausubel et al., eds., Greene Publishingand Wiley-Interscience, New York, 1995; and Tijssen, 1993, LaboratoryTechniques in Biochemistry and Molecular Biology: Hybridization withNucleic Acid Probes, Part I, Chapter 2, Elsevier, New York, 1993).Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent or highly stringent conditions to anucleotide sequence listed in Table 1 corresponds to a naturallyoccurring nucleic acid molecule.

There are a variety of methods that can be used to produce libraries ofpotential homologs from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene is then ligated into anappropriate expression vector. Use of a degenerate set of genes allowsfor the provision, in one mixture, of all of the sequences encoding thedesired set of potential sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (See, e.g., Narang, 1983,Tetrahedron 39:3; Itakura et al., 1984, Annu. Rev. Biochem. 53:323;Itakura et al., 1984, Science 198:1056; Ike et al., 1983, Nucleic AcidRes. 11:477).

Additionally, optimized nucleic acids can be created. Preferably, anoptimized nucleic acid encodes a polypeptide that has a function similarto those of the polypeptides listed in Table 1 and/or modulates aplant's growth and/or yield under normal and/or water-limited conditionsand/or tolerance to an environmental stress, and more preferablyincreases a plant's growth and/or yield under normal and/orwater-limited conditions and/or tolerance to an environmental stressupon its overexpression in the plant. As used herein, “optimized” refersto a nucleic acid that is genetically engineered to increase itsexpression in a given plant or animal. To provide plant optimizednucleic acids, the DNA sequence of the gene can be modified to: 1)comprise codons preferred by highly expressed plant genes; 2) comprisean A+T content in nucleotide base composition to that substantiallyfound in plants; 3) form a plant initiation sequence; 4) to eliminatesequences that cause destabilization, inappropriate polyadenylation,degradation and termination of RNA, or that form secondary structurehairpins or RNA splice sites; or 5) elimination of antisense openreading frames. Increased expression of nucleic acids in plants can beachieved by utilizing the distribution frequency of codon usage inplants in general or in a particular plant. Methods for optimizingnucleic acid expression in plants can be found in EPA 0359472; EPA0385962; PCT Application No. WO 91/16432; U.S. Pat. No. 5,380,831; U.S.Pat. No. 5,436,391; Perlack et al., 1991, Proc. Natl. Acad. Sci. USA88:3324-3328; and Murray et al., 1989, Nucleic Acids Res. 17:477-498.

An isolated polynucleotide of the invention can be optimized such thatits distribution frequency of codon usage deviates, preferably, no morethan 25% from that of highly expressed plant genes and, more preferably,no more than about 10%. In addition, consideration is given to thepercentage G+C content of the degenerate third base (monocotyledonsappear to favor G+C in this position, whereas dicotyledons do not). Itis also recognized that the XCG (where X is A, T, C, or G) nucleotide isthe least preferred codon in dicots, whereas the XTA codon is avoided inboth monocots and dicots. Optimized nucleic acids of this invention alsopreferably have CG and TA doublet avoidance indices closelyapproximating those of the chosen host plant. More preferably, theseindices deviate from that of the host by no more than about 10-15%.

The invention further provides an isolated recombinant expression vectorcomprising a polynucleotide as described above, wherein expression ofthe vector in a host cell results in the plant's increased growth and/oryield under normal or water-limited conditions and/or increasedtolerance to environmental stress as compared to a wild type variety ofthe host cell. The recombinant expression vectors of the inventioncomprise a nucleic acid of the invention in a form suitable forexpression of the nucleic acid in a host cell, which means that therecombinant expression vectors include one or more regulatory sequences,selected on the basis of the host cells to be used for expression, whichis operatively linked to the nucleic acid sequence to be expressed. Asused herein with respect to a recombinant expression vector,“operatively linked” is intended to mean that the nucleotide sequence ofinterest is linked to the regulatory sequence(s) in a manner whichallows for expression of the nucleotide sequence (e.g., in a bacterialor plant host cell when the vector is introduced into the host cell).The term “regulatory sequence” is intended to include promoters,enhancers, and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are well known in the art.Regulatory sequences include those that direct constitutive expressionof a nucleotide sequence in many types of host cells and those thatdirect expression of the nucleotide sequence only in certain host cellsor under certain conditions. It will be appreciated by those skilled inthe art that the design of the expression vector can depend on suchfactors as the choice of the host cell to be transformed, the level ofexpression of polypeptide desired, etc. The expression vectors of theinvention can be introduced into host cells to thereby producepolypeptides encoded by nucleic acids as described herein.

Plant gene expression should be operatively linked to an appropriatepromoter conferring gene expression in a timely, cell specific, ortissue specific manner. Promoters useful in the expression cassettes ofthe invention include any promoter that is capable of initiatingtranscription in a plant cell. Such promoters include, but are notlimited to, those that can be obtained from plants, plant viruses, andbacteria that contain genes that are expressed in plants, such asAgrobacterium and Rhizobium.

The promoter may be constitutive, inducible, developmentalstage-preferred, cell type-preferred, tissue-preferred, ororgan-preferred. Constitutive promoters are active under mostconditions. Examples of constitutive promoters include the CaMV 19S and35S promoters (Odell et al., 1985, Nature 313:810-812), the sX CaMV 35Spromoter (Kay et al., 1987, Science 236:1299-1302) the Sep1 promoter,the rice actin promoter (McElroy et al., 1990, Plant Cell 2:163-171),the Arabidopsis actin promoter, the ubiquitan promoter (Christensen etal., 1989, Plant Molec. Biol. 18:675-689), pEmu (Last et al., 1991,Theor. Appl. Genet. 81:581-588), the figwort mosaic virus 35S promoter,the Smas promoter (Velten et al., 1984, EMBO J 3:2723-2730), the superpromoter (U.S. Pat. No. 5,955,646), the GRP1-8 promoter, the cinnamylalcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), promoters fromthe T-DNA of Agrobacterium, such as mannopine synthase, nopalinesynthase, and octopine synthase, the small subunit of ribulosebiphosphate carboxylase (ssuRUBISCO) promoter, and the like.

Inducible promoters are preferentially active under certainenvironmental conditions, such as the presence or absence of a nutrientor metabolite, heat or cold, light, pathogen attack, anaerobicconditions, and the like. For example, the hsp80 promoter from Brassicais induced by heat shock; the PPDK promoter is induced by light; thePR-1 promoters from tobacco, Arabi-dopsis, and maize are inducible byinfection with a pathogen; and the Adh1 promoter is induced by hypoxiaand cold stress. Plant gene expression can also be facilitated via aninducible pro-moter (For a review, see Gatz, 1997, Annu. Rev. PlantPhysiol. Plant Mol. Biol. 48:89-108). Chemically inducible promoters areespecially suitable if gene expression is wanted to occur in a timespecific manner. Examples of such promoters are a salicylic acidinducible promoter (PCT Application No. WO 95/19443), a tetracyclineinducible promoter (Gatz et al., 1992, Plant J. 2: 397-404), and anethanol inducible promoter (PCT Application No. WO 93/21334).

In one preferred embodiment of the present invention, the induciblepromoter is a stress-inducible promoter. For the purposes of theinvention, stress-inducible promoters are preferentially active underone or more of the following stresses: sub-optimal conditions associatedwith salinity, drought, nitrogen, temperature, metal, chemical,pathogenic, and oxidative stresses. Stress inducible promoters include,but are not limited to, Cor78 (Chak et al., 2000, Planta 210:875-883;Hovath et al., 1993, Plant Physiol. 103:1047-1053), Cor15a (Artus etal., 1996, PNAS 93(23):13404-09), Rci2A (Medina et al., 2001, PlantPhysiol. 125:1655-66; Nylander et al., 2001, Plant Mol. Biol. 45:341-52;Navarre and Goffeau, 2000, EMBO J. 19:2515-24; Capel et al., 1997, PlantPhysiol. 115:569-76), Rd22 (Xiong et al., 2001, Plant Cell 13:2063-83;Abe et al., 1997, Plant Cell 9:1859-68; Iwasaki et al., 1995, Mol. Gen.Genet. 247:391-8), cDet6 (Lang and Palve, 1992, Plant Mol. Biol.20:951-62), ADH1 (Hoeren et al., 1998, Genetics 149:479-90), KAT1(Nakamura et al., 1995, Plant Physiol. 109:371-4), KST1 (Müller-Röber etal., 1995, EMBO 14:2409-16), Rha1 (Terryn et al., 1993, Plant Cell5:1761-9; Terryn et al., 1992, FEBS Lett. 299(3):287-90), ARSK1(Atkinson et al., 1997, GenBank Accession #L22302, and PCT ApplicationNo. WO 97/20057), PtxA (Plesch et al., GenBank Accession #X67427),SbHRGP3 (Ahn et al., 1996, Plant Cell 8:1477-90), GH3 (Liu et al., 1994,Plant Cell 6:645-57), the pathogen inducible PRP1-gene promoter (Ward etal., 1993, Plant. Mol. Biol. 22:361-366), the heat induciblehsp80-promoter from tomato (U.S. Pat. No. 5,187,267), cold induciblealpha-amylase promoter from potato (PCT Application No. WO 96/12814), orthe wound-inducible pinII-promoter (European Patent No. 375091). Forother examples of drought, cold, and salt-inducible promoters, such asthe RD29A promoter, see Yamaguchi-Shinozalei et al., 1993, Mol. Gen.Genet. 236:331-340.

Developmental stage-preferred promoters are preferentially expressed atcertain stages of development. Tissue and organ preferred promotersinclude those that are preferentially expressed in certain tissues ororgans, such as leaves, roots, seeds, or xylem. Examples oftissue-preferred and organ-preferred promoters include, but are notlimited to fruit-preferred, ovule-preferred, male tissue-preferred,seed-preferred, integument-preferred, tuber-preferred, stalk-preferred,pericarp-preferred, leaf-preferred, stigma-preferred, pollen-preferred,anther-preferred, petal-preferred, sepal-preferred, pedicel-preferred,silique-preferred, stem-preferred, root-preferred promoters, and thelike. Seed-preferred promoters are preferentially expressed during seeddevelopment and/or germination. For example, seed-preferred promoterscan be embryo-preferred, endosperm-preferred, and seed coat-preferred(See Thompson et al., 1989, BioEssays 10:108). Examples ofseed-preferred promoters include, but are not limited to, cellulosesynthase (celA), Cim1, gamma-zein, globulin-1, maize 19 kD zein(cZ19B1), and the like.

Other suitable tissue-preferred or organ-preferred promoters include thenapin-gene promoter from rapeseed (U.S. Pat. No. 5,608,152), theUSP-promoter from Vicia faba (Bae-umlein et al., 1991, Mol. Gen. Genet.225(3): 459-67), the oleosin-promoter from Arabidopsis (PCT ApplicationNo. WO 98/45461), the phaseolin-promoter from Phaseolus vulgaris (U.S.Pat. No. 5,504,200), the Bce4-promoter from Brassica (PCT ApplicationNo. WO 91/13980), or the legumin B4 promoter (LeB4; Baeumlein et al.,1992, Plant Journal, 2(2): 233-9), as well as promoters conferring seedspecific expression in monocot plants like maize, barley, wheat, rye,rice, etc. Suitable promoters to note are the Ipt2 or Ipt1-gene promoterfrom barley (PCT Application No. WO 95/15389 and PCT Application No. WO95/23230) or those described in PCT Application No. WO 99/16890(promoters from the barley hordein-gene, rice glutelin gene, rice oryzingene, rice prolamin gene, wheat gliadin gene, wheat glutelin gene, oatglutelin gene, Sorghum kasirin-gene, and rye secalin gene).

Other promoters useful in the expression cassettes of the inventioninclude, but are not limited to, the major chlorophyll a/b bindingprotein promoter, histone promoters, the Ap3 promoter, the β-conglycinpromoter, the napin promoter, the soybean lectin promoter, the maize 15kD zein promoter, the 22 kD zein promoter, the 27 kD zein promoter, theg-zein promoter, the waxy, shrunken 1, shrunken 2, and bronze promoters,the Zm13 promoter (U.S. Pat. No. 5,086,169), the maize polygalacturonasepromoters (PG) (U.S. Pat. Nos. 5,412,085 and 5,545,546), and the SGB6promoter (U.S. Pat. No. 5,470,359), as well as synthetic or othernatural promoters.

Additional flexibility in controlling heterologous gene expression inplants may be obtained by using DNA binding domains and responseelements from heterologous sources (i.e., DNA binding domains fromnon-plant sources). An example of such a heterologous DNA binding domainis the LexA DNA binding domain (Brent and Ptashne, 1985, Cell43:729-736).

In a preferred embodiment of the present invention, the polynucleotideslisted in Table 1 are expressed in plant cells from higher plants (e.g.,the spermatophytes, such as crop plants). A polynucleotide may be“introduced” into a plant cell by any means, including transfection,transformation or transduction, electroporation, particle bombardment,agroinfection, and the like. Suitable methods for transforming ortransfecting plant cells are disclosed, for example, using particlebombardment as set forth in U.S. Pat. Nos. 4,945,050; 5,036,006;5,100,792; 5,302,523; 5,464,765; 5,120,657; 6,084,154; and the like.More preferably, the transgenic corn seed of the invention may be madeusing Agrobacterium transformation, as described in U.S. Pat. Nos.5,591,616; 5,731,179; 5,981,840; 5,990,387; 6,162,965; 6,420,630, U.S.patent application publication number 2002/0104132, and the like.Transformation of soybean can be performed using for example a techniquedescribed in European Patent No. EP 0424047, U.S. Pat. No. 5,322,783,European Patent No. EP 0397 687, U.S. Pat. No. 5,376,543, or U.S. Pat.No. 5,169,770. A specific example of wheat transformation can be foundin PCT Application No. WO 93/07256. Cotton may be transformed usingmethods disclosed in U.S. Pat. Nos. 5,004,863; 5,159,135; 5,846,797, andthe like. Rice may be transformed using methods disclosed in U.S. Pat.Nos. 4,666,844; 5,350,688; 6,153,813; 6,333,449; 6,288,312; 6,365,807;6,329,571, and the like. Other plant transformation methods aredisclosed, for example, in U.S. Pat. Nos. 5,932,782; 6,153,811;6,140,553; 5,969,213; 6,020,539, and the like. Any plant transformationmethod suitable for inserting a transgene into a particular plant may beused in accordance with the invention.

According to the present invention, the introduced polynucleotide may bemaintained in the plant cell stably if it is incorporated into anon-chromosomal autonomous replicon or integrated into the plantchromosomes. Alternatively, the introduced polynucleotide may be presenton an extra-chromosomal non-replicating vector and may be transientlyexpressed or transiently active.

Another aspect of the invention pertains to an isolated polypeptidehaving a sequence selected from the group consisting of the polypeptidesequences listed in Table 1. An “isolated” or “purified” polypeptide isfree of some of the cellular material when produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemicallysynthesized. The language “substantially free of cellular material”includes preparations of a polypeptide in which the polypeptide isseparated from some of the cellular components of the cells in which itis naturally or recombinantly produced. In one embodiment, the language“substantially free of cellular material” includes preparations of apolypeptide of the invention having less than about 30% (by dry weight)of contaminating polypeptides, more preferably less than about 20% ofcontaminating polypeptides, still more preferably less than about 10% ofcontaminating polypeptides, and most preferably less than about 5%contaminating polypeptides.

The determination of activities and kinetic parameters of enzymes iswell established in the art. Experiments to determine the activity ofany given altered enzyme must be tailored to the specific activity ofthe wild-type enzyme, which is well within the ability of one skilled inthe art. Overviews about enzymes in general, as well as specific detailsconcerning structure, kinetics, principles, methods, applications andexamples for the determination of many enzyme activities are abundantand well known to one skilled in the art.

The invention is also embodied in a method of producing a transgenicplant comprising at least one polynucleotide listed in Table 1 or Table2, wherein expression of the polynucleotide in the plant results in theplant's increased growth and/or yield under normal and/or water-limitedconditions and/or increased tolerance to an environmental stress ascompared to a wild type variety of the plant comprising the steps of:(a) introducing into a plant cell an expression vector comprising atleast one polynucleotide listed in Table 1 or Table 2, and (b)generating from the plant cell a transgenic plant that expresses thepolynucleotide, wherein expression of the polynucleotide in thetransgenic plant results in the plant's increased growth and/or yieldunder normal or water-limited conditions and/or increased tolerance toenvironmental stress as compared to a wild type variety of the plant.The plant cell may be, but is not limited to, a protoplast, gameteproducing cell, and a cell that regenerates into a whole plant. As usedherein, the term “transgenic” refers to any plant, plant cell, callus,plant tissue, or plant part, that contains at least one recombinantpolynucleotide listed in Table 1 or Table 2. In many cases, therecombinant polynucleotide is stably integrated into a chromosome orstable extra-chromosomal element, so that it is passed on to successivegenerations.

The present invention also provides a method of increasing a plant'sgrowth and/or yield under normal and/or water-limited conditions and/orincreasing a plant's tolerance to an environmental stress comprising thesteps of increasing the expression of at least one polynucleotide listedin Table 1 or Table 2 in the plant. Expression of a polynucleotidelisted in Table 1 or Table 2 can be increased by any method known tothose of skill in the art.

The effect of the genetic modification on plant growth and/or yieldand/or stress tolerance can be assessed by growing the modified plantunder normal and/or less than suitable conditions and then analyzing thegrowth characteristics and/or metabolism of the plant. Such analysistechniques are well known to one skilled in the art, and include dryweight, wet weight, polypeptide synthesis, carbohydrate synthesis, lipidsynthesis, evapotranspiration rates, general plant and/or crop yield,flowering, reproduction, seed setting, root growth, respiration rates,photosynthesis rates, metabolite composition, etc., using methods knownto those of skill in bio-technology.

The invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof.

EXAMPLE 1 Identification of P. patens Open Reading Frames

cDNA libraries made from plants of the species P. patens (Hedw.) B.S.G.from the collection of the genetic studies section of the University ofHamburg were sequences using standard methods. The plants originatedfrom the strain 16/14 collected by H. L. K. Whitehouse in Gransden Wood,Huntingdonshire (England), which was subcultured from a spore by Engel(1968, Am. J. Bot. 55:438-446).

Sequences were processed and annotated using the software packageEST-MAX commercially provided by Bio-Max (Munich, Germany). The programincorporates practically all bioinformatics methods important forfunctional and structural characterization of protein sequences. Forreference, see the website at pedant.mips.biochem.mpg.de. The mostimportant algorithms incorporated in EST-MAX are: FASTA (Very sensitivesequence database searches with estimates of statistical significance;Pearson, 1990, Methods Enzymol. 183:63-98); BLAST (Very sensitivesequence database searches with estimates of statistical significance;Altschul et al., 1990, Journal of Molecular Biology 215:403-10);PREDATOR (High-accuracy secondary structure prediction from single andmultiple sequences; Frishman and Argos, 1997, Proteins 27:329-335);CLUSTALW (Multiple sequence alignment; Thompson et al., 1994, NucleicAcids Research 22:4673-4680); TMAP (Transmembrane region prediction frommultiply aligned sequences; Persson and Argos, 1994, J. Mol. Biol.237:182-192); ALOM2 (Transmembrane region prediction from singlesequences; Klein et al., 1984, Biochim. Biophys. Acta 787:221-226.Version 2 by Dr. K. Nakai); PROSEARCH (Detection of PROSITE proteinsequence patterns; Kolakowski et al., 1992, Biotechniques 13, 919-921);BLIMPS (Similarity searches against a database of ungapped blocks,Wallace and Henikoff, 1992, Comput Appl Biosci. 8(3):249-54); PATMAT (asearching and extraction program for sequence, pattern and block queriesand databases, CABIOS 8:249-254. Written by Bill Alford).

P. patens partial cDNAs (ESTs) were identified in the P. patens ESTsequencing program using the program EST-MAX through BLAST analysis. Thefull-length nucleotide cDNA sequences were determined using knownmethods. The identity and similarity of the amino acid sequences of thedisclosed polypeptide sequences to known protein sequences are shown inTables 2-19 (Pairwise Comparison was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum 62).

TABLE 3 Comparison of PpTPT-1 (SEQ ID NO: 2) to known RNA 2′phosphotransferases Public Database Sequence Sequence Accession #Species Identity (%) Similarity (%) XP_550615 O. sativa 45.4 56.2NP_197750 A. thaliana 34.1 43.0 NP_182058 A. thaliana 39.6 48.9NP_594515 Schizosaccharomyces 21.3 29.1 pombe NP_788477 Drosophila 25.534.7 melanogaster

TABLE 4 Comparison of PpCDC2-1 (SEQ ID NO: 4) to known Cell DivisionControl Protein 2 Public Database Sequence Sequence Accession # SpeciesIdentity (%) Similarity (%) S42049 Picea abies 92.9 96.3 Q40790 Pinuscontorta 92.5 96.3 CDC2_CHERU Chenopodium 91.5 95.9 rubrum Q9AUH4Populus tremula × 90.5 95.9 P. tremuloides Q8W2D3 Helianthus annuus 89.595.2

TABLE 5 Comparison of PpLRP-1 (SEQ ID NO: 6) to known Leucine RichRepeat Family Proteins Public Database Sequence Sequence Accession #Species Identity (%) Similarity (%) NP_912570 O. sativa 27.6 42.1NP_921136 O. sativa 27.2 40.3 AAF71805 A. thaliana 24.4 33.6 NP_177947A. thaliana 28.1 38.5 G96811 A. thaliana 28.8 40.3

TABLE 6 Comparison of PpRBP-1 (SEQ ID NO: 8) to known Ran bindingprotein 1 Public Database Sequence Sequence Accession # Species Identity(%) Similarity (%) Q94K24 Lycopersicon 50.7 63.3 esculentum Q9LUZ8 A.thaliana 39.4 51.9 NP_200667 A. thaliana 44.3 58.3 O04149 A. thaliana44.1 55.1 NP_172194 A thaliana 44.4 55.1

TABLE 7 Comparison of PpPD-1 (SEQ ID NO: 10) to known Plastid divisionftsZ proteins Public Database Sequence Sequence Accession # SpeciesIdentity (%) Similarity (%) Q70ZZ6 P. patens 100 100 Q75ZR3 Nannochloris42.6 53.0 bacillaris ZP_00177632 Crocosphaera watsonii 41.3 51.8NP_440816 Synechocystis sp. 41.0 52.1 T51092 Synechocystis sp. 40.0 51.5

TABLE 8 Comparison of PpMSC-1 (SEQ ID NO: 12) to known Mitochondiralsubstrate carrier proteins Public Database Sequence Sequence Accession #Species Identity (%) Similarity (%) NP_194188 A. thaliana 56.6 70.0T05577 A. thaliana 56.7 69.8 Q66PX4 Saccharum 55.4 69.9 officinarumNP_179836 A. thaliana 54.3 71.4 D84613 A. thaliana 54.5 71.2

TABLE 9 Comparison of PpMBP-1 (SEQ ID NO: 14) to known MADS-box proteinsPublic Database Sequence Sequence Accession # Species Identity (%)Similarity (%) Q8LPA5 P. patens 63.0 63.0 Q6QAF0 P. patens 62.6 62.6Q9FE71 P. patens 53.4 55.8 Q9FE89 P. patens 49.1 53.7 Q8LLC8 Lycopodium46.1 59.5 annotinum

TABLE 10 Comparison of PpAK-1 (SEQ ID NO: 16) to known Adenosine kinaseproteins Public Database Sequence Sequence Accession # Species Identity(%) Similarity (%) ADK_PHYPA P. patens 100 100 NP_195950 A. thaliana66.3 77.8 XP_466836 O. sativa 68.2 77.6 Q84P58 O. sativa 62.9 71.5NP_187593 A. thaliana 64.7 76.6

TABLE 11 Comparison of PpZF-6 (SEQ ID NO: 18) to known zinc fingerproteins Public Database Sequence Sequence Accession # Species Identity(%) Similarity (%) NP_180709 A. thaliana 61.1 71.0 XP_472997 O. sativa63.4 72.1 NP_176722 A. thaliana 61.8 72.4 T02366 A. thaliana 54.7 64.1NP_172080 A. thaliana 58.9 69.0

TABLE 12 Comparison of PpCDK-1 (SEQ ID NO: 20) to known Cyclin-dependentkinase regulatory subunits Public Database Sequence Sequence Accession #Species Identity (%) Similarity (%) Q6JJ57 Ipomoea 76.9 83.5 trifidaQ6T300 G. max 79.1 82.4 NP_180364 A. thaliana 74.7 82.4 XP_470214 O.sativa 80.2 84.6 Q8GZU5 Populus tremula × 75.8 80.2 P. tremuloides

TABLE 13 Comparison of PpZF-7 (SEQ ID NO: 22) to known RING zinc fingerproteins Public Database Sequence Sequence Accession # Species Identity(%) Similarity (%) Q6F3A0 O. sativa 34.7 47.3 Q852N7 O. sativa 35.5 48.5Q7XJB5 O. sativa 33.1 45.9 NP_851050 A. thaliana 35.3 46.2 NP_851051 A.thaliana 35.3 46.2

TABLE 14 Comparison of PpMFP-1 (SEQ ID NO: 24) to known MAR bindingfilament-like protein 1 Public Database Sequence Sequence Accession #Species Identity (%) Similarity (%) MFP1_TOBAC Nicotiana tabacum 18.030.5 NP_914440 O. sativa 16.8 30.1 MFP1_ARATH A. thaliana 19.2 30.3NP_188221 A. thaliana 20.1 31.1 T07111 Lycopersicon 19.6 31.6 esculentum

TABLE 15 Comparison of PpLRP-2 (SEQ ID NO: 26) to known Leucine richrepeat receptor-like protein kinases Public Database Sequence SequenceAccession # Species Identity (%) Similarity (%) Q9XGG1 Sorghum bicolor9.3 16.1 NP_189183 A. thaliana 9.2 14.6 Q708X5 Cicer arietinum 18.0 28.6XP_474976 O. sativa 5.9 10.3 Q9LSU7 A. thaliana 9.2 14.0

TABLE 16 Comparison of PpPPK-1 (SEQ ID NO: 28) to known light-sensorprotein kinases Public Database Sequence Sequence Accession # SpeciesIdentity (%) Similarity (%) S27396 Ceratodon purpureus 5.7 11.4 P93098Ceratodon purpureus 5.8 11.5 PHY1_CERPU Ceratodon purpureus 5.7 11.4NP_564829 A. thaliana 19.0 32.1 H96666 A. thaliana 19.5 31.6

TABLE 17 Comparison of PpSRP-1 (SEQ ID NO: 30) to Synaptobrevin-relatedproteins Public Database Sequence Sequence Accession # Species Identity(%) Similarity (%) NP_180871 A. thaliana 73.0 84.2 Q7X9C5 Pyruspyrifolia 64.9 75.2 NP_180826 A. thaliana 55.4 63.8 Q681H0 A. thaliana71.2 83.8

TABLE 18 Comparison of PpCBL-1 (SEQ ID NO: 32) to known Calcineurin Bproteins Public Database Sequence Sequence Accession # Species Identity(%) Similarity (%) XP_3133573 Anopheles gambiae 19.1 36.7 NP_505885Caenorhabditis 16.6 34.8 elegans Q95P81 Bombyx mori 18.7 36.4 NP_524874D. melanogaster 17.1 35.3

TABLE 19 Comparison of PpCBL-2 (SEQ ID NO: 34) to known caleosin-relatedproteins Public Database Sequence Sequence Accession # Species Identity(%) Similarity (%) Q9SQ57 Sesamum indicum 64.9 78.4 T07092 G. max 67.574.6 NP_194404 A. thaliana 61.2 74.7 NP_200335 A. thaliana 62.1 72.8XP_473140 O. sativa 60.2 73.0

TABLE 20 Comparison of PpHD-1 (SEQ ID NO: 36) to known histonedeacetylase proteins Public Database Sequence Sequence Accession #Species Identity (%) Similarity (%) Q8W508 Zea mays 81.9 88.6 NP_190054A. thaliana 78.7 88.2 T47443 A. thaliana 76.1 85.8 Q6JJ24 Ipomoeatrifida 68.2 75.6 Q7XLX3 Oryza sativa 70.1 77.5

EXAMPLE 2 Cloning of Full-Length cDNAs from Other Plants

Canola, soybean, rice, maize, linseed, and wheat plants were grown undera variety of conditions and treatments, and different tissues wereharvested at various developmental stages. Plant growth and harvestingwere done in a strategic manner such that the probability of harvestingall expressable genes in at least one or more of the resulting librariesis maximized. The mRNA was isolated from each of the collected samples,and cDNA libraries were constructed. No amplification steps were used inthe library production process in order to minimize redundancy of geneswithin the sample and to retain expression information. All librarieswere 3′ generated from mRNA purified on oligo dT columns. Colonies fromthe transformation of the cDNA library into E. coli were randomly pickedand placed into microtiter plates.

Plasmid DNA was isolated from the E. coli colonies and then spotted onmembranes. A battery of 288 ³³P radiolabeled 7-mer oligonucleotides weresequentially hybridized to these membranes. To increase throughput,duplicate membranes were processed. After each hybridization, a blotimage was captured during a phosphorimage scan to generate ahybridization profile for each oligonucleotide. This raw data image wasautomatically transferred to a computer. Absolute identity wasmaintained by barcoding for the image cassette, filter, and orientationwithin the cassette. The filters were then treated using relatively mildconditions to strip the bound probes and returned to the hybridizationchambers for another round of hybridization. The hybridization andimaging cycle was repeated until the set of 288 oligomers was completed.

After completion of the hybridizations, a profile was generated for eachspot (representing a cDNA insert), as to which of the 288 ³³Pradiolabeled 7-mer oligonucleotides bound to that particular spot (cDNAinsert), and to what degree. This profile is defined as the signaturegenerated from that clone. Each clone's signature was compared with allother signatures generated from the same organism to identify clustersof related signatures. This process “sorts” all of the clones from anorganism into clusters before sequencing.

The clones were sorted into various clusters based on their havingidentical or similar hybridization signatures. A cluster should beindicative of the expression of an individual gene or gene family. Aby-product of this analysis is an expression profile for the abundanceof each gene in a particular library. One-path sequencing from the 5′end was used to predict the function of the particular clones bysimilarity and motif searches in sequence databases.

The full-length DNA sequence of the P. patens PpHD-1 (SEQ ID NO:35) wasblasted against proprietary databases of canola, soybean, rice, maize,linseed, and wheat cDNAS at an e value of e⁻¹⁰ (Altschul et al., 1997,Nucleic Acids Res. 25: 3389-3402). All the contig hits were analyzed forthe putative full length sequences, and the longest clones representingthe putative full length contigs were fully sequenced. Two homologs fromcanola (BnHD-1, SEQ ID NO:38 and BnHD-2, SEQ ID NO:40), one homolog frommaize (ZmHD-1, SEQ ID NO:42), one homolog from linseed (LuHD-1, SEQ IDNO:44), one sequence from rice (OsHD-1, SEQ ID NO:46) three sequencesfrom soybean (GmHD-1, SEQ ID NO:48, GmHD-2, SEQ ID NO:50, and GmHD-3,SEQ ID NO:52) and one sequence from wheat (TaHD-1, SEQ ID NO:54) wereidentified. The degree of amino acid identity and similarity of thesesequences to the closest known public sequence is indicated in Table 21(Pairwise Comparison was used: gap penalty: 10; gap extension penalty:0.1; score matrix: blosum62).

TABLE 21 Degree of Amino Acid Identity and Similarity of HistoneDeacetylases Gene Name Public Database Sequence Sequence (SEQ ID NO)Accession # Species Identity (%) Similarity (%) BnHD-1 NP_190054 A.thaliana   96% 98.1% (SEQ ID NO: 38) BnHD-2 NP_201116 A. thaliana 92.6%95.3% (SEQ ID NO: 40) ZmHD-1 NP_563817 A. thaliana 66.4% 77.2% (SEQ IDNO: 42) LuHD-1 NP_190054 A. thaliana 87.4% 94.4% (SEQ ID NO: 44) OsHD-1Q7Y0Y8 O. sativa  100%  100% (SEQ ID NO: 46) GmHD-1 NP_563817 A.thaliana 63.1%   74% (SEQ ID NO: 48) GmHD-2 NP_190054 A. thaliana 85.6%92.1% (SEQ ID NO: 50) GmHD-2 NP_567921 A. thaliana 67.2% 77.4% (SEQ IDNO: 52) TaHD-1 Q7Y0Y8 O. sativa 89.4% 93.8% (SEQ ID NO: 54)

EXAMPLE 3 Stress-Tolerant Arabidopsis Plants

A fragment containing the P. patens polynucleotide was ligated into abinary vector containing a selectable marker gene. The resultingrecombinant vector contained the corresponding polynucleotide listed inTable 1 in the sense orientation under the constitutive super promoter.The recombinant vectors were transformed into Agrobacterium tumefaciensC58C1 and PMP90 plants according to standard conditions. A. thalianaecotype C24 plants were grown and transformed according to standardconditions. T1 plants were screened for resistance to the selectionagent conferred by the selectable marker gene, and T1 seeds werecollected.

The P. patens polynucleotides were overexpressed in A. thaliana underthe control of a constitutive promoter. T2 and/or T3 seeds were screenedfor resistance to the selection agent conferred by the selectable markergene on plates, and positive plants were transplanted into soil andgrown in a growth chamber for 3 weeks. Soil moisture was maintainedthroughout this time at approximately 50% of the maximum water-holdingcapacity of soil.

The total water lost (transpiration) by the plant during this time wasmeasured. After 3 weeks, the entire above-ground plant material wascollected, dried at 65° C. for 2 days and weighed. The ratio ofabove-ground plant dry weight (DW) to plant water use is water useefficiency (WUE). Tables 22-41 present WUE and DW for independenttransformation events (lines) of transgenic plants overexpressing the P.patens polynucleotides. Least square means (LSM), standard errors, andsignificant value (P) of a line compared to wild-type controls from anAnalysis of Variance are presented. The percent improvement of each P.patens polynucleotides line as compared to wild-type control plants forWUE and DW is also presented.

TABLE 22 A. thaliana lines overexpressing PpTPT-1 (SEQ ID NO: 2)Measure- Standard % ment Genotype Line LSM Error Improvement P DWWild-type 0.136 0.011 — — PpTPT-1 8 0.217 0.025 60 0.0033 (SEQ ID 60.222 0.028 64 0.0047 NO: 2) 5 0.226 0.032 67 0.0085 10 0.228 0.025 680.0009 2 0.230 0.032 70 0.0063 WUE Wild-type 2.270 0.085 — — PpTPT-1 82.274 0.190 0 0.9822 (SEQ ID 6 2.308 0.212 2 0.8656 NO: 2) 2 2.426 0.2457 0.5465 5 2.675 0.245 18 0.1207

TABLE 23 A. thaliana lines overexpressing PpCDC2-1 (SEQ ID NO: 4)Measure- Standard % ment Genotype Line LSM Error Improvement P DWWild-type 0.088 0.009 — — PpCDC2- 4 0.117 0.016 33 0.1147 1 1 0.1250.016 42 0.0473 (SEQ ID NO: 4) WUE Wild-type 1.446 0.097 — — PpCDC2- 41.890 0.186 31 0.036  1 1 1.947 0.186 35 0.0183 (SEQ ID NO: 4)

TABLE 24 A. thaliana lines overexpressing PpLRP-1 (SEQ ID NO: 6)Measure- Standard % ment Genotype Line LSM Error Improvement P DWWild-type 0.109 0.033 — — PpLRP-1 10 0.161 0.034 48 0.2835 (SEQ ID NO:6) WUE Wild-type 1.782 0.119 — — PpLRP-1 10 2.205 0.169 24 0.0529 (SEQID NO: 6)

TABLE 25 A. thaliana lines overexpressing PpRBP-1 (SEQ ID NO: 8)Measure- Standard % ment Genotype Line LSM Error Improvement P DWWild-type 0.088 0.008 — — PpRBP-1 2 0.130 0.017 48 0.0276 (SEQ ID NO: 8)WUE Wild-type 1.446 0.102 — — PpRBP-1 2 2.301 0.214 59 0.0004 (SEQ IDNO: 8)

TABLE 26 A. thaliana lines overexpressing PpPD-1 (SEQ ID NO: 10)Measure- Standard % ment Genotype Line LSM Error Improvement P DWWild-type 0.114 0.006 — — PpPD-1 3 0.171 0.019 50 0.0045 (SEQ ID 1 0.1710.017 50 0.0019 NO: 10) 8 0.174 0.019 53 0.0026 2 0.182 0.019 60 0.000711 0.191 0.017 67 <.0001 5 0.201 0.019 76 <.0001 9 0.204 0.017 79 <.0001WUE Wild-type 1.958 0.058 — — PpPD-1 11 2.328 0.165 19 0.0353 (SEQ ID 12.343 0.165 20 0.0286 NO: 10) 8 2.354 0.180 20 0.0383 2 2.450 0.180 250.0102 3 2.517 0.180 29 0.0036 5 2.552 0.180 30 0.002  9 2.572 0.165 310.0006

TABLE 27 A. thaliana lines overexpressing PpMSC-1 (SEQ ID NO: 12)Measure- Standard % ment Genotype Line LSM Error Improvement P DWWild-type 0.136 0.011 — — PpMSC-1 3 0.227 0.027 68 0.0026 (SEQ ID 20.240 0.025 77 0.0002 NO: 12) 1 0.247 0.025 82 <.0001 4 0.271 0.022 100 <.0001 WUE Wild-type 2.270 0.085 — — PpMSC-1 2 2.343 0.191  3 0.7268(SEQ ID 4 2.631 0.174 16 0.0654 NO: 12) 1 2.820 0.191 24 0.0097

TABLE 28 A. thaliana lines overexpressing PpMBP-1 (SEQ ID NO: 14)Measure- Standard % ment Genotype Line LSM Error Improvement P DWWild-type 0.110 0.005 — — PpMBP-1 1 0.154 0.017 39 0.0146 (SEQ ID NO:14) WUE Wild-type 1.620 0.066 — — PpMBP-1 1 2.144 0.209 32 0.0182 (SEQID NO: 14)

TABLE 29 A. thaliana lines overexpressing PpAK-1 (SEQ ID NO: 16)Measure- Standard % ment Genotype Line LSM Error Improvement P DWWild-type 0.136 0.011 — — PpAK-1 1 0.185 0.022 36 0.049  (SEQ ID 5 0.2160.022 59 0.0014 NO: 16) 3 0.217 0.027 60 0.0063 7 0.217 0.024 60 0.00268 0.227 0.024 68 0.0008 4 0.230 0.024 69 0.0006 WUE Wild-type 2.2700.084 — — PpAK-1 8 2.285 0.188 1 0.9394 (SEQ ID 7 2.358 0.188 4 0.6683NO: 16) 5 2.374 0.172 5 0.5851 3 2.377 0.211 5 0.6369 4 2.403 0.188 60.5191

TABLE 30 A. thaliana lines overexpressing PpZF-6 (SEQ ID NO: 18)Measure- Standard % ment Genotype Line LSM Error Improvement P DWWild-type 0.107 0.015 — — PpZF-6 4 0.131 0.018 22 0.3091 (SEQ ID NO: 18)WUE Wild-type 1.897 0.316 — — PpZF-6 4 2.026 0.371  7 0.7946 (SEQ ID NO:18)

TABLE 31 A. thaliana lines overexpressing PpCDK-1 (SEQ ID NO: 20)Measure- Standard % ment Genotype Line LSM Error Improvement P DWWild-type 0.088 0.009 — — PpCDK-1 7 0.148 0.017 69 0.0029 (SEQ ID NO:20) WUE Wild-type 1.446 0.102 — — PpCDK-1 7 1.963 0.195 36 0.0207 (SEQID NO: 20)

TABLE 32 A. thaliana lines overexpressing PpZF-7 (SEQ ID NO: 22)Measure- Standard % ment Genotype Line LSM Error Improvement P DWWild-type 0.114 0.006 — — PpZF-7 1 0.151 0.019 32 0.0617 (SEQ ID 100.153 0.019 35 0.0456 NO: 22) 2 0.159 0.019 39 0.0226 7 0.160 0.019 400.0198 3 0.163 0.019 43 0.0139 6 0.175 0.019 54 0.0021 9 0.176 0.019 550.0018 5 0.177 0.019 56 0.0014 8 0.196 0.019 72 <.0001 4 0.217 0.019 90<.0001 WUE Wild-type 1.958 0.057 — — PpZF-7 2 2.185 0.176 12 0.2224 (SEQID 10 2.237 0.176 14 0.1331 NO: 22) 7 2.242 0.176 15 0.1262 9 2.3270.176 19 0.0479 3 2.359 0.176 20 0.0318 6 2.378 0.176 21 0.0245 1 2.4350.176 24 0.0108 5 2.490 0.176 27 0.0045 8 2.537 0.176 30 0.002  4 2.7070.176 38 <.0001

TABLE 33 A. thaliana lines overexpressing PpMFP-1 (SEQ ID NO: 24)Measure- Standard % ment Genotype Line LSM Error Improvement P DWWild-type 0.110 0.005 — — PpMFP-1 4 0.128 0.016 16 0.3008 (SEQ ID 20.130 0.016 18 0.25  NO: 24) 10 0.145 0.016 31 0.0465 3 0.146 0.016 320.0417 6 0.159 0.016 44 0.0048 7 0.164 0.016 48 0.0022 5 0.166 0.016 500.0015 1 0.168 0.016 52 0.0011 8 0.172 0.016 56 0.0004 WUE Wild-type1.620 0.064 — — PpMFP-1 3 1.979 0.203 22 0.0929 (SEQ ID 8 2.049 0.203 260.0451 NO: 24) 7 2.049 0.203 26 0.0449 4 2.095 0.203 29 0.0267 1 2.1130.203 30 0.0215 6 2.178 0.203 34 0.0094 5 2.217 0.203 37 0.0055 10 2.3240.203 43 0.0011 2 2.345 0.203 45 0.0008

TABLE 34 A. thaliana lines overexpressing PpLRP-2 (SEQ ID NO: 26)Measure- Standard % ment Genotype Line LSM Error Improvement P DWWild-type 0.136 0.011 — — PpLRP-2 4 0.199 0.029 47 0.042  (SEQ ID 20.206 0.023 52 0.0078 NO: 26) 3 0.224 0.029 65 0.0049 1 0.227 0.023 670.0007 5 0.235 0.026 74 0.0006 8 0.266 0.040 96 0.0026 WUE Wild-type2.270 0.090 — — PpLRP-2 4 2.360 0.224 4 0.7073 (SEQ ID 5 2.402 0.200 60.5481 NO: 26) 1 2.404 0.183 6 0.5095 8 2.471 0.317 9 0.5426

TABLE 35 A. thaliana lines overexpressing PpPPK-1 (SEQ ID NO: 28)Measure- Standard % ment Genotype Line LSM Error Improvement P DWWild-type 0.108 0.007 — — PpPPK-1 4 0.157 0.020 45 0.023  (SEQ ID 20.159 0.018 47 0.0097 NO: 28) 10 0.175 0.020 62 0.0018 9 0.177 0.022 640.0037 WUE Wild-type 1.951 0.078 — — PpPPK-1 4 2.043 0.219  5 0.6913(SEQ ID 9 2.158 0.245 11 0.4225 NO: 28) 2 2.177 0.200 12 0.2948 10 2.5230.219 29 0.0149

TABLE 36 A. thaliana lines overexpressing PpSRP-1 (SEQ ID NO: 30)Measure- Standard % ment Genotype Line LSM Error Improvement P DWWild-type 0.114 0.006 — — PpSRP-1 7 0.152 0.018 33 0.0495 (SEQ ID 60.159 0.018 39 0.0196 NO: 30) 1 0.162 0.020 42 0.026  10 0.164 0.017 440.0054 9 0.167 0.015 46 0.0015 8 0.174 0.018 53 0.0019 2 0.179 0.018 570.0008 WUE Wild-type 1.958 0.057 — — PpSRP-1 10 2.109 0.161  8 0.3776(SEQ ID 8 2.197 0.177 12 0.1991 NO: 30) 9 2.239 0.149 14 0.0802 2 2.3020.177 18 0.0659 6 2.405 0.177 23 0.017  1 2.450 0.197 25 0.0178

TABLE 37 A. thaliana lines overexpressing PpCBL-1 (SEQ ID NO: 32)Measure- Standard % ment Genotype Line LSM Error Improvement P DWWild-type 0.114 0.006 — — PpCBL-1 5 0.156 0.019 37 0.034  (SEQ ID 80.163 0.019 43 0.0153 NO: 32) 4 0.179 0.019 57 0.0012 3 0.180 0.019 580.0011 6 0.181 0.017 59 0.0003 1 0.182 0.017 59 0.0003 9 0.214 0.017 87<.0001 WUE Wild-type 1.958 0.057 — — PpCBL-1 3 2.109 0.177  8 0.4181(SEQ ID 5 2.152 0.177 10 0.2991 NO: 32) 8 2.158 0.177 10 0.2836 1 2.2980.162 17 0.0489 6 2.306 0.162 18 0.0438 9 2.319 0.162 18 0.0367 4 2.4400.177 25 0.0105

TABLE 38 A. thaliana lines overexpressing PpCBL-2 (SEQ ID NO: 34)Measure- Standard % ment Genotype Line LSM Error Improvement P DWWild-type 0.114 0.006 — — PpCBL-2 9 0.156 0.017 37 0.0226 (SEQ ID 100.170 0.017 49 0.0027 NO: 34) 1 0.179 0.017 57 0.0005 4 0.188 0.017 65<.0001 7 0.188 0.017 65 <.0001 3 0.192 0.017 68 <.0001 8 0.194 0.017 70<.0001 2 0.203 0.017 78 <.0001 WUE Wild-type 1.958 0.054 — — PpCBL-2 91.944 0.168 −1 0.9357 (SEQ ID 2 2.314 0.168 18 0.0451 NO: 34) 10 2.3220.168 19 0.0405 8 2.448 0.168 25 0.0061 3 2.545 0.168 30 0.0011 4 2.5690.168 31 0.0007 1 2.617 0.168 34 0.0003 7 2.771 0.168 42 <.0001

TABLE 39 A. thaliana lines overexpressinq PpHD-1 (SEQ ID NO: 36)Measure- Standard % ment Genotype Line LSM Error Improvement P DWWild-type 1.620 0.065 — — PpHD-1 7 1.976 0.207 22 0.1027 (SEQ ID 3 1.9850.207 23 0.0944 NO: 36) 6 2.144 0.207 32 0.0169 2 2.374 0.207 47 0.00078 2.444 0.207 51 0.0002 WUE Wild-type 0.110 0.005 — — PpHD-1 2 0.1260.016 14 0.3655 (SEQ ID 8 0.143 0.016 30 0.0566 NO: 36) 6 0.149 0.016 350.0246 3 0.152 0.016 37 0.0177 7 0.191 0.016 73 <.0001

TABLE 40 A. thaliana lines overexpressing PpLRP-1 (SEQ ID NO: 56)Measure- Standard % ment Genotype Line LSM Error Improvement P DWWild-type 0.109 0.033 — — PpLRP-1 10 0.161 0.034 48 0.2835 (SEQ ID NO:56) WUE Wild-type 1.782 0.119 — — PpLRP-1 10 2.205 0.169 24 0.0529 (SEQID NO: 56)

TABLE 41 A. thaliana lines overexpressing PpLRP-1 (SEQ ID NO: 58)Measure- Standard % ment Genotype Line LSM Error Improvement P DWWild-type 0.109 0.033 — — PpLRP-1 10 0.161 0.034 48 0.2835 (SEQ ID NO:58) WUE Wild-type 1.782 0.119 — — PpLRP-1 10 2.205 0.169 24 0.0529 (SEQID NO: 58)

EXAMPLE 4 Stress-Tolerant Rapeseed/Canola Plants

Canola cotyledonary petioles of 4 day-old young seedlings are used asexplants for tissue culture and transformed according to EP1566443. Thecommercial cultivar Westar (Agriculture Canada) is the standard varietyused for transformation, but other varieties can be used. A. tumefaciensGV3101:pMP90RK containing a binary vector is used for canolatransformation. The standard binary vector used for transformation ispSUN (WO 02/00900), but many different binary vector systems have beendescribed for plant transformation (e.g. An, G. in AgrobacteriumProtocols, Methods in Molecular Biology vol 44, pp 47-62, Gartland KMAand MR Davey eds. Humana Press, Totowa, N.J.). A plant gene expressioncassette comprising a selection marker gene and a plant promoterregulating the transcription of the cDNA encoding the polynucleotide isemployed. Various selection marker genes can be used including themutated acetohydroxy acid synthase (AHAS) gene disclosed in U.S. Pat.Nos. 5,767,366 and 6,225,105. A suitable promoter is used to regulatethe trait gene to provide constitutive, developmental, tissue orenvironmental regulation of gene transcription.

Canola seeds are surface-sterilized in 70% ethanol for 2 min, incubatedfor 15 min in 55° C. warm tap water and then in 1.5% sodium hypochloritefor 10 minutes, followed by three rinses with sterilized distilledwater. Seeds are then placed on MS medium without hormones, containingGamborg B5 vitamins, 3% sucrose, and 0.8% Oxoidagar. Seeds aregerminated at 24° C. for 4 days in low light (<50 μMol/m²s, 16 hourslight). The cotyledon petiole explants with the cotyledon attached areexcised from the in vitro seedlings, and inoculated with Agrobacteriumby dipping the cut end of the petiole explant into the bacterialsuspension. The explants are then cultured for 3 days on MS mediumincluding vitamins containing 3.75 mg/I BAP, 3% sucrose, 0.5 g/I MES, pH5.2, 0.5 mg/l GA3, 0.8% Oxoidagar at 24° C., 16 hours of light. Afterthree days of co-cultivation with Agrobacterium, the petiole explantsare transferred to regeneration medium containing 3.75 mg/l BAP, 0.5mg/l GA3, 0.5 g/l MES, pH 5.2, 300 mg/l timentin and selection agentuntil shoot regeneration. As soon as explants start to develop shoots,they are transferred to shoot elongation medium (A6, containing fullstrength MS medium including vitamins, 2% sucrose, 0.5% Oxoidagar, 100mg/l myo-inositol, 40 mg/l adenine sulfate, 0.5 g/l MES, pH 5.8, 0.0025mg/l BAP, 0.1 mg/l IBA, 300 mg/l timentin and selection agent).

Samples from both in vitro and greenhouse material of the primarytransgenic plants (TO) are analyzed by qPCR using TaqMan probes toconfirm the presence of T-DNA and to determine the number of T-DNAintegrations.

Seed is produced from the primary transgenic plants by self-pollination.The second-generation plants are grown in greenhouse conditions andself-pollinated. The plants are analyzed by qPCR using TaqMan probes toconfirm the presence of T-DNA and to determine the number of T-DNAintegrations. Homozygous transgenic, heterozygous transgenic and azygous(null transgenic) plants are compared for their stress tolerance, forexample, in the assays described in Example 3, and for yield, both inthe greenhouse and in field studies.

EXAMPLE 5 Screening for Stress-Tolerant Rice Plants

Transgenic rice plants comprising a polynucleotide of the invention aregenerated using known methods. Approximately 15 to 20 independenttransformants (TO) are generated. The primary transformants aretransferred from tissue culture chambers to a greenhouse for growing andharvest of T1 seeds. Five events of the T1 progeny segregated 3:1 forpresence/absence of the transgene are retained. For each of theseevents, 10 T1 seedlings containing the transgene (hetero- andhomozygotes), and 10 T1 seedlings lacking the transgene (nullizygotes)are selected by visual marker screening. The selected T1 plants aretransferred to a greenhouse. Each plant receives a unique barcode labelto link unambiguously the phenotyping data to the corresponding plant.The selected T1 plants are grown on soil in 10 cm diameter pots underthe following environmental settings: photoperiod=11.5 h, daylightintensity=30,000 lux or more, daytime temperature=28° C. or higher,night time temperature=22° C., relative humidity=60-70%. Transgenicplants and the corresponding nullizygotes are grown side-by-side atrandom positions. From the stage of sowing until the stage of maturity,the plants are passed several times through a digital imaging cabinet.At each time point digital, images (2048×1536 pixels, 16 millioncolours) of each plant are taken from at least 6 different angles.

The data obtained in the first experiment with T1 plants are confirmedin a second experiment with T2 plants. Lines that have the correctexpression pattern are selected for further analysis. Seed batches fromthe positive plants (both hetero- and homozygotes) in T1 are screened bymonitoring marker expression. For each chosen event, the heterozygoteseed batches are then retained for T2 evaluation. Within each seedbatch, an equal number of positive and negative plants are grown in thegreenhouse for evaluation.

Transgenic plants are screened for their improved growth and/or yieldand/or stress tolerance, for example, using the assays described inExample 3, and for yield, both in the greenhouse and in field studies.

EXAMPLE 6 Stress-Tolerant Soybean Plants

The polynucleotides of Tables 1 and 2 are transformed into soybean usingthe methods described in commonly owned copending internationalapplication number WO 2005/121345, the contents of which areincorporated herein by reference. The transgenic plants are thenscreened for their improved growth under water-limited conditions and/ordrought, salt, and/or cold tolerance, for example, using the assaysdescribed in Example 3, and for yield, both in the greenhouse and infield studies.

EXAMPLE 7 Stress-Tolerant Wheat Plants

Transformation of wheat is performed with the method described by Ishidaet al., 1996, Nature Biotech. 14745-50. Immature embryos areco-cultivated with Agrobacterium tumefaciens that carry “super binary”vectors, and transgenic plants are recovered through organogenesis. Thisprocedure provides a transformation efficiency between 2.5% and 20%. Thetransgenic plants are then screened for their improved growth and/oryield under water-limited conditions and/or stress tolerance, forexample, is the assays described in Example 3, and for yield, both inthe greenhouse and in field studies.

EXAMPLE 8 Stress-Tolerant Corn Plants

Agrobacterium cells harboring the genes and the maize ahas gene on thesame plasmid are grown in YP medium supplemented with appropriateantibiotics for 1-3 days. A loop of Agrobacterium cells is collected andsuspended in 1.5 ml M-LS-002 medium (LS-inf) and the tube containingAgrobacterium cells is kept on a shaker for 1-4 hours at 1,000 rpm.

Corncobs [genotype J553×(HIIIAxA188)] are harvested at 7-12 days afterpollination. The cobs are sterilized in 20% Clorox solution for 15minutes followed by thorough rinse with sterile water. Immature embryoswith size 0.8-2.0 mm are dissected into the tube containingAgrobacterium cells in LS-inf solution.

Agro-infection is carried out by keeping the tube horizontally in thelaminar hood at room temperature for 30 minutes. Mixture of the agroinfection is poured on to a plate containing the co-cultivation medium(M-LS-011). After the liquid agro-solution is piped out, the embryostransferred to the surface of a filter paper that is placed on the agarco-cultivation medium. The excess bacterial solution is removed with apipette. The embryos are placed on the co-cultivation medium withscutellum side up and cultured in the dark at 22° C. for 2-4 days.

Embryos are transferred to M-MS-101 medium without selection. Seven toten days later, embryos are transferred to M-LS-401 medium containing0.50 μM imazethapyr and grown for 4 weeks (two 2-week transfers) toselect for transformed callus cells. Plant regeneration is initiated bytransferring resistant calli to M-LS-504 medium supplemented with 0.75μM imazethapyr and grown under light at 25-27° C. for two to threeweeks. Regenerated shoots are then transferred to rooting box withM-MS-618 medium (0.5 μM imazethapyr). Plantlets with roots aretransferred to potting mixture in small pots in the greenhouse and afteracclimatization are then transplanted to larger pots and maintained ingreenhouse till maturity.

The copy number of the transgene in each plantlet is assayed usingTaqman analysis of genomic DNA, and transgene expression is assayedusing qRT-PCR of total RNA isolated from leaf samples.

Using assays such as the assay described in Example 3, each of theseplants is uniquely labeled, sampled and analyzed for transgene copynumber. Transgene positive and negative plants are marked and pairedwith similar sizes for transplanting together to large pots. Thisprovides a uniform and competitive environment for the transgenepositive and negative plants. The large pots are watered to a certainpercentage of the field water capacity of the soil depending theseverity of water-stress desired. The soil water level is maintained bywatering every other day. Plant growth and physiology traits such asheight, stem diameter, leaf rolling, plant wilting, leaf extension rate,leaf water status, chlorophyll content and photosynthesis rate aremeasured during the growth period. After a period of growth, the aboveground portion of the plants is harvested, and the fresh weight and dryweight of each plant are taken. A comparison of the drought tolerancephenotype between the transgene positive and negative plants is thenmade.

Using assays such as the assay described in Example 3, the pots arecovered with caps that permit the seedlings to grow through but minimizewater loss. Each pot is weighed periodically and water added to maintainthe initial water content. At the end of the experiment, the fresh anddry weight of each plant is measured, the water consumed by each plantis calculated and WUE of each plant is computed. Plant growth andphysiology traits such as WUE, height, stem diameter, leaf rolling,plant wilting, leaf extension rate, leaf water status, chlorophyllcontent and photosynthesis rate are measured during the experiment. Acomparison of WUE phenotype between the transgene positive and negativeplants is then made.

Using assays such as the assay described in Example 3, these pots arekept in an area in the greenhouse that has uniform environmentalconditions, and cultivated optimally. Each of these plants is uniquelylabeled, sampled and analyzed for transgene copy number.

The plants are allowed to grow under theses conditions until they reacha predefined growth stage. Water is then withheld. Plant growth andphysiology traits such as height, stem diameter, leaf rolling, plantwilting, leaf extension rate, leaf water status, chlorophyll content andphotosynthesis rate are measured as stress intensity increases. Acomparison of the dessication tolerance phenotype between transgenepositive and negative plants is then made.

Segregating transgenic corn seeds for a transformation event are plantedin small pots for testing in a cycling drought assay. These pots arekept in an area in the greenhouse that has uniform environmentalconditions, and cultivated optimally. Each of these plants is uniquelylabeled, sampled and analyzed for transgene copy number. The plants areallowed to grow under theses conditions until they reach a predefinedgrowth stage. Plants are then repeatedly watered to saturation at afixed interval of time. This water/drought cycle is repeated for theduration of the experiment. Plant growth and physiology traits such asheight, stem diameter, leaf rolling, leaf extension rate, leaf waterstatus, chlorophyll content and photosynthesis rate are measured duringthe growth period. At the end of the experiment, the plants areharvested for above-ground fresh and dry weight. A comparison of thecycling drought tolerance phenotype between transgene positive andnegative plants is then made.

In order to test segregating transgenic corn for drought tolerance underrain-free conditions, managed-drought stress at a single location ormultiple locations is used. Crop water availability is controlled bydrip tape or overhead irrigation at a location which has less than 10 cmrainfall and minimum temperatures greater than 5° C. expected during anaverage 5 month season, or a location with expected in-seasonprecipitation intercepted by an automated “rain-out shelter” whichretracts to provide open field conditions when not required. Standardagronomic nomic practices in the area are followed for soil preparation,planting, fertilization and pest con-trol. Each plot is sown with seedsegregating for the presence of a single transgenic insertion event. ATaqman transgene copy number assay is used on leaf samples todifferentiate the transgenics from null-segregant control plants. Plantsthat have been genotyped in this manner are also scored for a range ofphenotypes related to drought-tolerance, growth and yield. Thesephenotypes include plant height, grain weight per plant, grain numberper plant, ear number per plant, above ground dry-weight, leafconductance to water vapor, leaf CO₂ uptake, leaf chlorophyll content,photosynthesis-related chlorophyll fluorescence parameters, water useefficiency,leaf water potential, leaf relative water content, stem sapflow rate, stem hydraulic conductivity, leaf temperature, leafreflectance, leaf light absorptance, leaf area, days to flowering,anthesis-silking interval, duration of grain fill, osmotic potential,osmotic adjustment, root size, leaf extension rate, leaf angle, leafrolling and survival. All measurements are made with commerciallyavailable instrumentation for field physiology, using the standardprotocols provided by the manufacturers. Individual plants are used asthe replicate unit per event.

In order to test non-segregating transgenic corn for drought toleranceunder rain-free conditions, managed-drought stress at a single locationor multiple locations is used. Crop water availability is controlled bydrip tape or overhead irrigation at a location which has less than 10 cmrainfall and minimum temperatures greater than 5° C. expected during anaverage 5 month season, or a location with expected in-seasonprecipitation intercepted by an automated “rain-out shelter” whichretracts to provide open field conditions when not required. Standardagronomic practices in the area are followed for soil preparation,planting, fertilization and pest control. Trial layout is designed topair a plot containing a non-segregating transgenic event with anadjacent plot of null-segregant controls. A null segregant is progeny(or lines derived from the progeny) of a transgenic plant that does notcontain the transgene due to Mendelian segregation. Additionalreplicated paired plots for a particular event are distributed aroundthe trial. A range of phenotypes related to drought-tolerance, growthand yield are scored in the paired plots and estimated at the plotlevel. When the measurement technique could only be applied toindividual plants, these are selected at random each time from withinthe plot. These pheno-types include plant height, grain weight perplant, grain number per plant, ear number per plant, above grounddry-weight, leaf conductance to water vapor, leaf CO₂ uptake, leafchlorophyll content, photosynthesis-related chlorophyll fluorescenceparameters, water use efficiency, leaf water potential, leaf relativewater content, stem sap flow rate, stem hydraulic conductivity, leaftemperature, leaf reflectance, leaf light absorptance, leaf area, daysto flowering, anthesis-silking interval, duration of grain fill, osmoticpotential, osmotic adjustment, root size, leaf extension rate, leafangle, leaf rolling and survival. All measurements are made withcommercially available instrumentation for field physiology, using thestandard protocols provided by the manufacturers. Individual plots areused as the replicate unit per event.

To perform multi-location testing of transgenic corn for droughttolerance and yield, five to twenty locations encompassing major corngrowing regions are selected. These are widely distributed to provide arange of expected crop water availabilities based on averagetemperature, humidity, precipitation and soil type. Crop wateravailability is not modified beyond standard agronomic practices. Triallayout is designed to pair a plot containing a non-segregatingtransgenic event with an adjacent plot of null-segregant controls. Arange of pheno-types related to drought-tolerance, growth and yield arescored in the paired plots and estimated at the plot level. When themeasurement technique could only be applied to individual plants, theseare selected at random each time from within the plot. These phenotypesincluded plant height, grain weight per plant, grain number per plant,ear number per plant, above ground dry-weight, leaf conductance to watervapor, leaf CO₂ uptake, leaf chlorophyll content,photo-synthesis-related chlorophyll fluorescence parameters, water useefficiency, leaf water potential, leaf relative water content, stem sapflow rate, stem hydraulic conductivity, leaf temperature, leafreflectance, leaf light absorptance, leaf area, days to flowering,anthesis-silking interval, duration of grain fill, osmotic potential,osmotic adjustment, root size, leaf extension rate, leaf angle, leafrolling and survival. All measurements are made with commerciallyavailable instru-mentation for field physiology, using the standardprotocols provided by the manufacturers. Individual plots are used asthe replicate unit per event.

1. A transgenic plant cell or a transgenic plant transformed with anexpression cassette comprising an isolated polynucleotide encoding apolypeptide having a sequence comprising amino acids 1 to 91 of SEQ IDNO:20.
 2. An isolated polynucleotide having a sequence selected from thegroup consisting of (a) a polynucleotide having a sequence comprisingnucleotides 248 to 520 of SEQ ID NO:19; and (b) a polynucleotide whichencodes a polypeptide having a sequence comprising amino acids 1 to 91of SEQ ID NO:20.
 3. An isolated polypeptide having a sequence comprisingamino acids 1 to 91 of SEQ ID NO:20.
 4. A method of producing atransgenic plant comprising the steps of: (a) introducing into a plantcell an expression vector comprising at least one polynucleotide havinga sequence selected from the group consisting of (i) a polynucleotidehaving a sequence comprising nucleotides 248 to 520 of SEQ ID NO:19 and(ii) a polynucleotide which encodes a polypeptide having a sequencecomprising amino acids 1 to 91 of SEQ ID NO:20; and (b) generating fromthe plant cell a transgenic plant that expresses the polynucleotide. 5.A method of increasing a plant's growth or yield under normal orwater-limited conditions or increasing a plant's tolerance to anenvironmental stress comprising the steps of: (a) inserting into anexpression vector a polynucleotide having a sequence selected from thegroup consisting of (i) a polynucleotide having a sequence comprisingnucleotides 248 to 520 of SEQ ID NO:19 and (ii) a polynucleotide whichencodes a polypeptide having a sequence comprising amino acids 1 to 91of SEQ ID NO:20; and (b) introducing the expression vector into theplant.